WO2023034849A2 - HIGH MOLECULAR WEIGHT MODIFIED dsRNA COMPOSITIONS - Google Patents
HIGH MOLECULAR WEIGHT MODIFIED dsRNA COMPOSITIONS Download PDFInfo
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- WO2023034849A2 WO2023034849A2 PCT/US2022/075740 US2022075740W WO2023034849A2 WO 2023034849 A2 WO2023034849 A2 WO 2023034849A2 US 2022075740 W US2022075740 W US 2022075740W WO 2023034849 A2 WO2023034849 A2 WO 2023034849A2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/60—Isolated nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P23/00—Chemosterilants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/02—Acaricides
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/04—Insecticides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2330/00—Production
- C12N2330/30—Production chemically synthesised
Definitions
- insects are usually civilization’s most important competitors for food, fiber, and other natural resources. Insects have a direct impact on agricultural food production by chewing the leaves of crop plants, sucking out plant juices, boring within the roots, stems or leaves, and spreading plant pathogens. Insects feed on natural fibers, destroy wooden building materials, ruin stored grain, and accelerate the process of decay.
- RNA interference is a naturally occurring biological process by which doublestranded ribonucleic acid (dsRNA) silences (knocks down) target gene expression in a sequence specific manner.
- Cellular enzymes use dsRNA to target and cleave single stranded RNA (ssRNA), including messenger RNA (mRNA) and non-coding RNA.
- ssRNA single stranded RNA
- mRNA messenger RNA
- RNAi is known to occur in many eukaryotes, including plants, insects, acari, fungi and animals, and offers great potential for selective and efficient regulation of gene expression.
- the dsRNA has an antisense strand containing sequence complementary to a sequence in the mRNA or non-coding RNA and a sense strand containing sequence complementary to the guide strand sequence and substantially identical to the sequence in the mRNA or non-coding RNA.
- the sense and antisense sequences can be present on separate RNA strands or on a single strand. When present on a single strand, the complementary sequences are connected by a non-hybridizing hairpin or loop sequence.
- RNAi-mediated gene suppression on targeted plants, insects, acari, and fungi affecting crops described in the prior art has been achieved using exogenously supplied unmodified dsRNA (UdsRNA) (US Patent 9,121,022; Ivashuta et al. 2015; US Publication No. 20160215290; Koch et al. 2016). It has been found, that when dsRNAs are used to induce RNAi in insects and are supplied in the insects’ diet, 60 base pair (bp) or longer dsRNAs are sometimes required for efficient uptake and processing (B perfumesi et al. 2012).
- UdsRNA unmodified dsRNA
- UdsRNA longer than about 30 base pairs (bp) has been achieved by in vitro transcription (Timmons 2006) and by fermentation (Fire et al. 1998).
- Commercially feasible large-scale methods for preparation and purification of the UdsRNA has been described (Arhancet et al., US9822361B2).
- UdsRNAs are sensitive to degradation by nucleases in the environment and the host, reducing efficacy of inhibition of gene expression (Baum 2016).
- RNAi triggers RNAi triggers
- siRNAs targeting insects using nucleotides chemically modified at the 2'-OH position of the ribose have been also described (Gong et al. 2013)
- the cost and synthetic complexity of modified siRNAs is neither economically feasible or sufficiently scalable, for preparation of amounts larger than a few grams, or of chemically modified dsRNA longer than about 30 bp.
- Post-transcriptional chemical modification of ssRNA for analytical purposes was also described by Merino (2005).
- dsRNA reacted more than 80 times less efficiently, with less than 0.18% of nucleotides in a stem region being modified and only within one (1) nucleotide of the end of a stem (e.g. within one nucleotide of a single strand region) Similar results have been observed for reaction of RNA with other reactants (Nodin 2015).
- l-methyl-7-nitroisatoic anhydride (1M7), benzoyl cyanide (BzCN), 2-methyl-3 -furoic acid imidazolide (FAI), and 2-methylnicotinic acid imidazolide (NAI) have been used to post- transcriptionally produce 2'-ribose esters of RNA, but have a similarly low percentage of the modification, with modification primarily occurring at riboses of unpaired nucleotides or their immediately adjacent paired nucleotides.
- Unmodified poly-ribonucleic acid is an exceptionally unstable molecule. Unlike DNA, it contains a hydroxyl group at the 2' position of the ribose which renders the RNA polymer sensitive to hydrolysis. Deprotonation of 2'-OH and, consequently, nucleophilic attack of 2' oxygen on the backbone phosphorus is the primary molecular mechanism of cleavage of phosphodiester bonds by various nucleolytic enzymes, including RNase A (Elliot and Ladomery, 2011). Stabilization of the nucleic acid backbone against nucleolysis has become a success-defining issue, much the same as for the evolution of siRNA-based therapeutics (Nair et al., 2014).
- RNA stability is especially important in intestinal milieu rich in nucleolytic activity with various endonucleases and phosphodiesterases responsible for digestion of nucleic acids (Whitt and Savage, 1988; Liu et al., 2015).
- a highly effective strategy for stabilizing RNA molecules is the derivatization of 2'-0 group.
- Various modifications at this position have been demonstrated to improve enzymatic stability of various RNA molecules, including nanoassemblies (Liu et al. 2010) and RNAi silencing molecules (Khvorova and Watts, 2017). These modifications are generally applied during chemical synthesis of small interfering RNAs (siRNAs).
- MdsRNAs post-transcriptionally chemically modified double strand RNAs having a high molecular weight polyalkyloxy modification at the 2’- OH position. This modification allows for greater bioavailability of the compound, better stability of the compound, and allows for greater stability against nucleases. As described herein, these MdsRNAs can be economically produced in a readily scalable manner.
- compositions of modified double strand RNA having chemically modified nucleotides, such that the MdsRNAs are modified to contain high molecular weight polyalkyloxy polymers, optionally further comprising 2’-0 chemically modified nucleotides to contain low molecular weight (LMW) moi eties (LMW modified nucleotides).
- LMW low molecular weight
- This disclosure also describes synthetic methods for efficiently making these modified MdsRNAs.
- compositions comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein B and R 1 are defined herein.
- MdsRNA post-transcriptionally chemically modified double strand RNA
- the post-transcriptional chemical modification of the double strand RNA comprises no more than about 30% of all the nucleotides being modified with high molecular weight polyalkyloxy polymers. In another embodiment, the post-transcriptional chemical modification of the double strand RNA further comprises at least about 2% of all the nucleotides being modified with LMW moieties. In one embodiment, the post-transcriptional chemical modification of the double strand RNA further comprises from about 2% to about 50% of all the nucleotides being modified with LMW moieties.
- the post-transcriptional chemical modification of the double strand RNA comprises about a 3:5:2 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 3:6.5:0.5 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post- transcriptional chemical modification of the double strand RNA comprises about a 1 :7:2 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides.
- the post-transcriptional chemical modification of the double strand RNA comprises about a 0.5:9:0.5 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.3:4:5.7 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.01 :4:5.99 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.06:0:96.4 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides.
- the disclosure provides a method of preparing a composition comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein B and R 1 are defined herein.
- MdsRNA post-transcriptionally chemically modified double strand RNA
- the post- transcriptional chemical modification of the double strand RNA comprises no more than about 30% of all the nucleotides being modified with high molecular weight polyalkyloxy polymers.
- the post- transcriptional chemical modification of the double strand RNA further comprises at least about 2% of all the nucleotides being modified with LMW moieties.
- the post-transcriptional chemical modification of the double strand RNA further comprises from about 2% to about 50% of all the nucleotides being modified with LMW moieties.
- the post-transcriptional chemical modification of the double strand RNA comprises about a 3:5:2 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In another embodiment of the methods of preparing compositions, the post- transcriptional chemical modification of the double strand RNA comprises about a 3:6.5:0.5 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides.
- the post-transcriptional chemical modification of the double strand RNA comprises about a 1 :7:2 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment of the methods of preparing compositions, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.5:9:0.5 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides.
- the post-transcriptional chemical modification of the double strand RNA comprises about a 0.3:4:5.7 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment of the methods of preparing compositions, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.01 :4:5.99 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides.
- the post-transcriptional chemical modification of the double strand RNA comprises about a 0.06:0:96.4 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides.
- the disclosure provides methods of modifying the expression of polynucleotides of interest in an insect, a fungus, a weed or an acari, using any of the compositions, target insects, fungi, weeds or acari, and sequences set forth herein.
- FIG. 1 shows cumulative mortality for Diamondback moth larvae after administration of select compounds of Formula (I).
- FIG. 2 shows decrease of dsRNA content on cabbage leaves in the field for treatment C2, unmodified dsSNF7, and modified dsSNF7 treatments NS2 (PEG-dsSNF7) and NS5 (NMIA-dsSNF7).
- FIG. 3 shows the mortality rate of Diamondback moth field larvae after administration of select compounds of Formula (I) after 3-days (3 DA-A) and 4-days (4 DA-A).
- FIG. 4 shows Agarose gel electrophoresis analysis of 300 bp dsRNA modified with activated 10k PEG. Lines: 1 - DNA ladder; 2 - original dsRNA; 3 - lOkPEG- MdsRNA reaction mixture.
- the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term ' about ' is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
- sequence or “nucleotide sequence” refers to a succession or order of nucleobases, nucleotides, and/or nucleosides, described with a succession of letters using the standard nucleotide nomenclature and the key for modified nucleotides described herein.
- sequences as described herein are listed from the 5’ terminus to the 3’ terminus.
- a MdsRNA is at least 40, at least 30, at least 50, at least 70, at least 80, at least 90, or at least 100 base pairs in length.
- a MdsRNA sense strand contains a sense sequence and a MdsRNA antisense strand contains an antisense sequence.
- the antisense sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary or at least 80% (partial) complementary to a nucleotide sequence present in a target gene transcribed mRNA or non-coding RNA (i.e., expressed RNA).
- the sense sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary or at least 80% (partially) complementary the antisense sequence.
- a sense sequence may also be 100% identical, at least 90% identical, or at least 80% identical to a nucleotide sequence (target sequence) present in a target gene mRNA or non-coding RNA.
- the sense sequence and a corresponding antisense sequence are partially (at least 80%), substantially (90%), or fully (100%) complementary to each other.
- the region of complementarity (antisense sequence) or identity (sense sequence) between the MdsRNA and a corresponding sequence in the target gene transcribed mRNA or noncoding RNA sequence is greater than 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length.
- the antisense sequence contains a contiguous sequence greater than 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length that is 100% complementary or at least 80% complementary to a corresponding contiguous sequence in the target gene transcribed mRNA or non-coding RNA.
- MdsRNA sense and antisense sequences can be either the same length or they can be 60 different lengths. Suitable sense and antisense sequences are identified using known methods readily available in the art.
- nucleotide refers to one base pairing unit (e.g., a unit of Formula I or an unmodified ribose) comprising a purine or pyrimidine base pairing moiety.
- the term may refer to the nucleotide unit with or without the attached intersubunit linkage, although, when referring to a “charged subunit”, the charge typically resides within the intersubunit linkage.
- the purine or pyrimidine base pairing moiety also referred to herein simply as a “nucleobases,” “base,” or “bases,” may be adenine, cytosine, guanine, uracil, thymine or inosine.
- bases such as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6- alkylpyrimidines (e.g.
- 6-methyluridine 6-methyluridine
- propyne quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5'- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, P-D- galactosylqueosine, 1 -methyladenosine, 1 -methylinosine, 2,2-dimethylguanosine, 3- methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7- methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5- methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio- N6-isopentenyladenosine, 13-D-mannosylqueo
- modified bases in this aspect is meant nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as illustrated above; such bases can be used at any position in the antisense molecule.
- Ts and Us are interchangeable. For instance, with other antisense chemistries such as 2'-O- methyl antisense oligonucleotides that are more RNA-like, the T bases may be shown as U.
- a “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the nucleotides in a MdsRNA are post-synthetically modified. Modified nucleotides include, but are not limited to, nucleotides having a ribose 2'-OH substitution.
- modified nucleotides can each have the same modification or they can have different modifications from each other, selected from high molecular weight polyalkyloxy polymers, and if present, low molecular weight moieties.
- dsRNA is an RNA molecule that has not been chemically modified.
- polyalkoxy or “polyalkyloxy” as used interchangeably herein refers to a suitable water-soluble polymer characterized by repeating alkoxy units.
- PEG polyethylene glycol
- PPG polypropylene glycol
- poloxamers hyaluronic acid
- polyvinyl alcohols polyoxazolines
- polyanhydrides poly(ortho esters)
- polycarbonates polyurethanes
- polyacrylic acids polyacrylamides
- polyacrylates polymethacrylates, polyorganophosphazenes
- polysiloxanes polyvinylpyrrolidone
- polycyanoacrylates polyesters, or any derivatives of the foregoing.
- the term “polyalkoxy” or “polyalkyloxy” used herein refers to a suitable linear or branched polyethylene glycol (PEG) polymer.
- the polyalkoxy is a linear or branched polypropylene glycol (PPG) polymer.
- the polyalkyloxy is a poloxamer.
- the polymer is an ethylene glycol-propylene glycol block copolymer poloxamer.
- the polymer is a poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol), PEG-PPG-PEG poloxamer.
- the polymer is an ethylene oxi de-propylene oxide triblock copolymer.
- the polyalkyloxy has a molecular weight of at least about 400 Da, at least about 1 kDa, at least about 5 kDa, or at least about 10 kDa. In another embodiment, the molecular weight of the polyalkyloxy is between about 400 Da and about 40 kDa or between about 5 kDa and about 40 kDa.
- polystyrene resin refers to nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene.
- poloxamers are poloxamers 407, 338, 188, 184, and 401 (i.e., F127, F108, L68, L64 and L121 Pluronic®, BASF). The first two digits multiplied by 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit multiplied by 10 gives the percentage polyoxyethylene content. “L” stands for liquid and “F” stands for flake solid.
- the amphiphilic character, that is the presence of both hydrophilic and lipophilic groups, of the resulting polymer modified dsRNAs of the disclosure modulate their physical and chemical properties such as solubility, absorption and permeability through plant tissue and target cell membranes and resistance to nucleases.
- the polymers that produced the most efficacious MdsRNAs have Hydrophilic-Lipohilic Balance (HLB) numbers ranging from 8 to 27. In one embodiment, the HLB is from about 2 to about 30. In another embodiment, the HLB is from about 15 to about 27.
- HLB Hydrophilic-Lipohilic Balance
- the term “high molecular weight polyalkyloxy” refers to a compound, substituent, or other chemical moiety comprising a polyalkyloxy group having a molecular weight of at least about 400 Da.
- the molecular weight of the polyalkyloxy is at least about 1 kDa.
- the molecular weight of the polyalkyloxy is at least about 5 kDa.
- the molecular weight of the polyalkyloxy is at least about 10 kDa.
- the molecular weight of the polyalkyloxy is between about 400 Da and about 40 kDa.
- the molecular weight of the polyalkyloxy is between about 5 kDa and about 40 kDa.
- a “low molecular weight (LMW) modified nucleotide” is a nucleotide having a 2’ -OH low molecular weight modification.
- Low molecular weight modifications are moi eties that are 1,000 daltons or less.
- R 2 in Formula III examples include, but are not limited to, C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5-C12 heteroaryl, wherein any of these is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or C1-C12 alkoxy (i.e., R 2 as described herein).
- At least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the nucleotides in a MdsRNA are post-transcriptionally chemically modified as LMW modified nucleotides.
- the chemical modifications are esters of N-m ethyl-anthranilic acid (from modification with N-methylisatoic anhydride (NMIA)), esters of N-benzyl-anthranilic acid (from modifications with N-benzylisatoic anhydride (NBIA)), dimethyl furoyl, esters of a fatty acid (e.g., Cl -Cl 8, such as but not limited to lauryl, oleic, linoleic), acetic acid, propionic acid, esters of amino acids (e.g., tyrosine, tryptophan, leucine), low molecular weight PEG, nitrogen containing moieties.
- NMIA N-methylisatoic anhydride
- NBIA N-benzyl-anthranilic acid
- dimethyl furoyl esters of a fatty acid (e.g., Cl -Cl 8, such as but not limited to lauryl, oleic, l
- the presence of the LMW moieties allows the composition to better dissolve in organic solvents, for delivery of the compositions to plants and ultimately to the target insects.
- the compositions can be made more concentrated, in amounts that are economical for the end-user.
- the decomposition rate in the field for the compositions of the application has been shown to decrease by filling in the spaces in the MdsRNA that are not modified with the high molecular polyalkyloxy polymers.
- the nucleotides that are not modified with high molecular weight polyalkyloxy polymer can be modified with low molecular weight moieties, as described herein.
- alkyl or “alkyl group” as used herein describes a univalent group derived from alkanes by removal of a hydrogen atom from any carbon atom -CnEbn+i.
- An alkyl group can be straight or branched chain or cyclic and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.
- a lower alkyl group contains from 1 to 25 carbon atoms in the principal chain.
- alkenyl as used herein are acyclic branched or unbranched hydrocarbons having one carbon-carbon double bond and the general formula -CnEbn-i. One or more of the hydrogen atoms can be substituted.
- An alkyl group can be straight or branched chain and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.
- an alkenyl contains from 2 to 25 carbon atoms in the principal chain.
- alkoxide or “alkoxy” as used herein is the conjugate base of an alcohol.
- the alcohol can be straight chain, branched, cyclic, and includes aryloxy compounds and include methoxy, ethoxy, isoproyloxy, butoxy, and the like.
- alkynyl as used herein are acyclic branched or unbranched hydrocarbons having a carbon-carbon triple bond and the general formula -CnH2n-3. They can be straight or branched chain and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like. As used herein a lower alkynyl containing from 2 to 25 carbon atoms in the principal chain.
- aryl or “Ar” as used herein alone or as part of another group denote optionally substituted homocyclic aromatic groups.
- Aryl groups can be monocyclic or bicyclic groups containing from 5 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl, or substituted naphthyl.
- heteroaryl as used herein alone or as part of another group denotes optionally substituted aromatic groups having at least one heteroatom in at least one ring.
- heteroaromatic group contains 5 or 6 atoms in each ring.
- a heteroaromatic group contains 1 or 2 oxygen atoms and/or 1 to 4 nitrogen atoms in the ring, and is bonded to the remainder of the molecule through a carbon.
- Exemplary groups include furyl, benzofuryl, oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, benzoxadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl, carbazolyl, purinyl, quinolinyl, isoquinolinyl, imidazopyridyl, and the like.
- substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal, carbamyl, carbocyclo, cyano, ester, ether, halogen, heterocyclo, hydroxyl, keto, ketal, phosphor, nitro, and thio.
- heteroatom refers to atoms other than carbon and hydrogen.
- halogen refers to as used herein alone or as part of another group refer to chlorine, bromine, fluorine or iodine.
- greater stability refers to the compounds of the instant application in comparison to unmodified dsRNAs or MdsRNAs modified with low molecular weight polyalkyloxy. In some embodiments, greater stability means increased persistence in agricultural fields, in plant tissue, on plant leaves, etc. In some embodiments, greater stability means increased in vivo half-life. In some embodiments, greater stability means enhanced resistance to physiological conditions. In other embodiments, greater stability means enhanced resistance to nucleases.
- acceptable salts refers to salts derived from suitable inorganic and organic acids and inorganic and organic bases that are, within the scope of sound judgment, suitable for use in contact with the tissues of humans, lower animals, and plants without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
- a salt of a compound of the present disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
- the term “scalable” refers to the processes ability to produce quantities of the target chemical in commercially relevant amounts.
- the process of the instant application may allow for the production of compounds in the hundreds of milligram scale.
- the process of the instant application may allow for the production of compounds in the gram scale.
- the process of the instant application may allow for the production in the kilogram scale.
- the process of the instant application may allow for the production in the metric ton scale.
- activation agent refers to a compound that increases the nucleophilicity or electrophilicity of target moiety.
- the change in electron density can be the result of an ionic or covalent bond from the activating group.
- the activation agents described herein increase the electrophilicity of a target carbonyl.
- Non-limiting examples of activation agents are lewis acids, protons, and coupling reagents.
- Non-limiting examples of coupling reagents are benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (2-(lH- benzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate, Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU), N,N' -Dicyclohexylcarbodiimide (DCC), carbonyldiimidazole (CDI), and 1- [Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, Hexafluorophosphate Azabenzo
- suitable leaving group refers to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage.
- the leaving group helps facilitate the reaction by lowering the energy of activation.
- suitable leaving groups are halides, tosylated or mesylated alcohols, pseudohalides, amines, heterocycles, and the like.
- the suitable leaving group is imidazole.
- anhydrous solvent refers to a solvent that has been physically or chemically treated to reduce the water content of the solvent.
- Anhydrous solvents may contain less than about 1% by weight water, less than about 0.3% by weight water, or less than about 0.1% by weight water.
- MdsRNAs are described having no more than about 30% of all the ribose rings chemically modified at the 2'-OH position with high molecular weight polyalkyloxy polymers, and optionally at least about 2% of all the nucleotides being chemically modified with LMW modified moieties.
- the MdsRNAs are capable of inhibiting gene expression in a sequence specific manner, such as through RNA interference or antisense mechanisms.
- composition comprising a post- transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I):
- MdsRNA post- transcriptionally chemically modified double strand RNA
- B is a nucleobase
- R 1 is selected from: wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12- 1000, b is an integer from 12-1000, and c is an integer from 12-1000.
- compositions comprising a post- transcriptionally chemically modified double strand RNA (MdsRNA) wherein the
- MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
- B is a nucleobase
- R 1 is selected from:
- y is an integer from 1-8
- x is an integer from 12-1000
- a is an integer from 12-
- b is an integer from 12-1000, and c is an integer from 12-1000; and optionally wherein at least about 2% of all the nucleotides independently comprise
- B is a nucleobase
- R 2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5-C12 heteroaryl, wherein R 2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or Ci-C 12 alkoxy.
- R 1 is selected from:
- x is an integer from 80-1000.
- x is an integer from 50-900.
- a is an integer from 80-1000.
- b is an integer from 80-1000.
- c is an integer from 80-1000.
- R 1 has a molecular weight between 5,000 and 15,000 Da.
- R 1 has a molecular weight between 5,000 and 40,000 Da.
- R 1 is polyethylene glycol (PEG).
- R 1 is poloxamer 407, 338, 188, 184, 401 or any combination thereof.
- a is 101
- b is 56
- c is 101
- R 1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R 1 is a HMW polymer with an HLB ranging from about 15 to about 27.
- R 2 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- NMA N-methyl anthranoyl
- NBA N-benzyl anthranoyl
- dimethyl furoyl -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- lauroyl In an embodiment, lauroyl.
- R 1 is and R 2 is linoleyl.
- the ratio of Formula (I) to Formula (III) to unmodified nucleotides is about 3:5:2; 3:6.5:0.5; 1 :7:2; .05:9:0.5; 1.3:4:5.7; 0.01 :4:5.99; 0.06:0:96.4.
- from about 2% to about 50% of all nucleotides are modified with LMW moieties.
- the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect.
- an efficacious sequence or target gene useful in the products and methods of the disclosure will inhibit the expression of the target gene, act on the mid gut of the insect and increase mortality or induce growth stunting, or stop instar development.
- the target insect is Diamondback moth (Plutella xylostella), Gypsy moth, Red imported fire ant (Solenopsis invicta), Fall armyworm, Colorado potato beetle, Canola flea beetle, Aedes aegypti, or Western corn root worm (Diabrotica virgifera virgifera).
- the target insect is Pea aphid (Acyrthosiphon pisum), Soybean aphid, Piezodorus guildinii.
- the target is an acari, such as Verroa mite.
- the target is a weed, such as Palmer amaranth.
- the target is a fungus such as Palmer amaranth, Fusarium graminearum (Gibberella zeae) or Botrytis.
- the MdsRNA used in the compositions of the disclosure will be a nucleotide sequence that can inhibit the expression of target genes or regions in these insects.
- the MdsRNA comprises a sequence complementary to a target region in Diamondback moth, such as but not limited to AChE2, P450, P450 CYP6BFlvl, Cytokine receptor DOMELESS, DOUX, Protein MESH transcript variant XI, Venom carboylesterase-6 and VPASE-E.
- the MdsRNA comprises a sequence complementary to a target region in Fall armyworm, such as but not limited to P450 CYP9A58, Cytokine receptor DOMELESS, Dredd, VPASE, Protein MESH transcript variant XI, P450 CYP321 A8, P450 CYP6B2- like.
- the MdsRNA comprises a sequence complementary to a target region in Western corn root worm, such as but not limited to SNF7.
- the MdsRNA comprises a sequence selected from one of SEQ ID Nos. 1-269 and its reverse complementary strand. In a particular embodiment, the sequence is selected from one of SEQ ID Nos. 13 or 248-251 and its reverse complementary strand.
- the target insect is a Lepidopteran and can be targeted using any one of SEQ ID Nos. 1-269. In an embodiment, the Lepidopteran is targeted using any one of SEQ ID Nos. 13 or 248-251.
- composition comprising a post- transcriptionally modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
- MdsRNA post- transcriptionally modified double strand RNA
- B is a nucleobase
- R 1 is a linear or branched polyalkyloxy having a molecular weight between about 400 Da and about 15 kDa; optionally wherein at least about 2% of all the nucleotides independently comprise LMW modified nucleotides of Formula (III): or an acceptable salt thereof, wherein:
- B is a nucleobase
- R 2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5- C12 heteroaryl, wherein R 2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or Ci-C 12 alkoxy.
- R 1 is a linear or branched polyalkyloxy or a poloxamer having a molecular weight between about 1 kDa and about 15 kDa.
- R 1 is a linear or branched polyalkyloxy or a poloxamer having a molecular weight between about 5 kDa and about 15 kDa. [0085] In another embodiment, R 1 is a linear or branched polyalkyloxy or a poloxamer having a molecular weight between about 5 kDa and about 10 kDa.
- R 1 is a polyethylene glycol polymer.
- R 1 is a poloxamer.
- the poloxamer is a triblock polymer comprising poly(ethylene glycol)-block-poly(propylene glycol)-block- poly (ethylene glycol).
- R 1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R 1 is a HMW polymer with an HLB ranging from about 15 to about 27.
- R 2 is C1-C25 alkyl.
- the C1-C25 alkyl is substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or C1-C12 alkoxy.
- the C1-C25 alkyl is substituted with one, two, or three substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or C1-C12 alkoxy.
- R 2 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- NMA N-methyl anthranoyl
- NBA N-benzyl anthranoyl
- dimethyl furoyl -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- the ratio of Formula (I) to Formula (III) to unmodified nucleotides is about 3:5:2; 3:6.5:0.5; 1 :7:2; .05:9:0.5; 1.3:4:5.7; 0.01 :4:5.99; 0.06:0:96.4.
- from about 2% to about 50% of all nucleotides are modified with LMW moieties.
- the MdsRNA comprises a sequence complementary to an expressed RNA in target insects, weeds, fungi or acari, as discussed herein, the sequence listing and examples of this disclosure.
- y is an integer from 1-8
- x is an integer from 12-1000
- a is an integer from 12-1000
- b is an integer from 12-1000
- c is an integer from 12-1000.
- R 1 is selected from: wherein at least 2% of all the nucleotides independently comprise LMW modified nucleotides of Formula (III) as defined above.
- x is an integer from 80-1000.
- x is an integer from 50-900.
- a is an integer from 80-1000.
- b is an integer from 80-1000.
- c is an integer from 80-1000.
- R 1 has a molecular weight between 5,000 and 15,000 Da.
- R 1 has a molecular weight between 5,000 and 40,000 Da.
- R 1 is polyethylene glycol (PEG).
- R 1 is poloxamer 407, 338, 188, 184, 401, or any combination thereof.
- a is 101
- b is 56
- c is 101
- R 1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R 1 is a HMW polymer with an HLB ranging from about 15 to about 27.
- R 2 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- NMA N-methyl anthranoyl
- NBA N-benzyl anthranoyl
- dimethyl furoyl -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- the ratio of Formula (I) to Formula (III) to unmodified nucleotides is about 3:5:2; 3:6.5:0.5; 1 :7:2; .05:9:0.5; 1.3:4:5.7; 0.01 :4:5.99; 0.06:0:96.4.
- from about 2% to about 50% of all nucleotides are modified with LMW moieties.
- sequence of the MdsRNA is selected from one of 13 or 248-251.
- the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect, weed, fungi, acari or in any of the targets as discussed herein and the sequence listing and examples of this disclosure.
- the compound of Formula (I) is or an acceptable salt thereof, wherein each variable is defined above.
- the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above.
- the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above.
- the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above.
- the terminal -H can be substituted with -CH3.
- the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above.
- the terminal -H can be substituted with -CH3.
- the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above.
- the terminal -H can be substituted with -CH3.
- the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above.
- the terminal -H can be substituted with -CH3.
- each B of Formula I is the same.
- each B of Formula I is different.
- each R 1 of Formula I is the same.
- each R 1 of Formula I is different.
- the base pairs in the MdsRNA is at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 350, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1,000 in length.
- the base pairs in the MdsRNA is between about 40 base pairs and about 1,000 base pairs.
- the MdsRNA is between about 50 base pairs and about 900 base pairs.
- the MdsRNA is between about 70 base pairs and about 800 base pairs.
- the MdsRNA is between about 80 base pairs and about 700 base pairs.
- the MdsRNA is between about 90 base pairs and about 600 base pairs. In an embodiment, the MdsRNA is between about 100 nucleotides and about 500 nucleotides. In another embodiment, the MdsRNA is between about 200 base pairs and about 400 base pairs.
- a target sequence useful in the products and methods of this disclosure will be selected to target the lowest guanine-cytosine (GC) content, such as no more than about 60% and preferably about 40% or less of the GC content of the target insect, fungus or weed, to allow for the highest level of modification under mild reaction conditions.
- the MdsRNA targets a sequence with a low guanine-cytosine content.
- the guanine-cytosine content is no more than about 50%.
- the guanine-cytosine content is about 40% or less.
- the guanine-cytosine content is between about 30% and about 40%.
- guanine-cytosine content is about 30%, about 35%, about 40%, about 50% or about 55%.
- the degree of modification of the nucleotides will depend upon the GC content of the dsRNA.
- the degree of modification e.g., the percentage of nucleotides that can be modified
- the reaction conditions are such that the sense and antisense strands in the dsRNA are only partially dissociated (i.e., only partially unzipped), for example at temperatures below 60°C, or at low concentrations of a solvent or co-solvent capable of dissociating the sense and antisense strands, e.g., at DMSO concentrations below 70%, or at temperatures below 60°C and at lower than 70% DMSO
- the riboses in the nucleotides at positions located less than 10 contiguous nucleotides away from the beginning or the end of a perfectly paired stretch of dsRNA may be preferentially modified, i.e., they may be modified to a larger extent than the riboses in nucleotides located more than 10 contiguous nucleotides away.
- MdsRNA Post transcriptionally modified double strand RNA
- MdsRNA compounds are significantly less susceptible to degradation by nucleases in the environment and the host. MdsRNA down-regulate expression of polynucleotides present in the target host by the RNAi mechanism.
- MdsRNA compounds consist of UdsRNA in which some of the H atoms of its 2’OH groups have been replaced with different chemical moieties, for example with, benzoyl, lauroyl, oleyl, linoleyl, N-methyl anthranoyl, nicotinoyl, or furoyl (US Patent 10,131,911).
- MdsRNAs are expected to be similarly non-toxic as UdsRNA.
- a MdsRNA sense strand is connected to the antisense strand.
- a sense strand may be connected to an antisense strand via a non-hybridizing hairpin or loop sequence.
- a loop sequence can be about 4 to about 100 or more nucleotides in length. In some embodiments, a loop is 150 or more nucleotides in length (Hauge et al. 2009).
- a MdsRNA further comprises one or more additional sequences including, but not limited to: promoter sequences, 5' sequences, 3' sequences, terminator sequences, and polyA sequences.
- a promoter is a region (sequence) of DNA that initiates transcription of a gene.
- a promoter can be a bacterial promoter, archaea promoter, eukaryotic promoter, or a Pol I, Pol II, or Poll III promoter.
- a bacterial promoter comprises the sequence 5'-TTGACA-3' about 35 units upstream from the transcription start site and the sequence 5'-TATAAT-3' about 10 bp upstream from the transcription start site.
- Particular embodiments of the current disclosure call for higher molecular polyalkyloxy polymers to be covalently bound to the 2’ -OH position of the intersubunit linkages.
- Preferred embodiments of the disclosure call for a low percentage of the overall subunits to be substituted with such a polymer (e.g., less than about 30%).
- the percentage of the overall subunits to be substituted with such a polymer is about 1%.
- the percentage of substituted subunits is about 1% to about 30%.
- the percentage of substituted subunits is about 1% to about 5%.
- the percentage of substituted subunits is less than about 1%. In another embodiment, the percentage of substituted subunits is between about 0.1% and about 1%.
- the compounds of the disclosure show equal or superior properties to compounds having lower weight polyalkyloxy polymers at the 2’ -OH position.
- the compounds of the disclosure show equal or superior properties to compounds having lower weight polyalkyloxy polymer compounds because the instant compounds have better solubility, enhanced bioavailability, and are resistant to nucleases.
- PEG and similar groups are known to prevent crossing of membranes, the instant compounds readily cross the cell-membrane.
- the low percentage levels (e.g., about 1% to about 30%) of the polyalkyloxy polymers of the disclosure still impart the beneficial stability properties of other compounds with significantly higher percentages of PEG or PEG-type substitutions (e.g., the instant compounds are resistant to exonucleases at about 1% to about 30%).
- the compounds of the disclosure are resistant to hydrolysis by carboxylate esterase. This may be due to the polyalkyloxy polymers being too sterically large for the catalytic pocket of the esterase.
- the larger polyalkyloxy polymers may further hydrogen bond with the 2’ OH groups of the unmodified nucleotides, thus creating further steric hinderance, even at the low levels of modification as described herein.
- the modifications of the MdsRNA strands, as described herein may preferentially occur at the ends of the MdsRNA strands due to the steric hinderance of the molecules.
- the MdsRNA strands are resistant to exonucleases while still being available to endonucleases, allowing the compounds of the disclosure to enter into the RNAi process easier.
- compositions methods of making the compositions, nucleotide sequences and uses for target insects, fungi, weeds or acari are intended to be covered herein in this disclosure.
- the sequence is Plutella xylostella acetylcholinesterase 2 mRNA, GenBank AY061975.1 nucleotide #s: 512-811; catatcgga ggattgcctc tatttgaaca tatgggtgcc gcagcacttg cgcgtccgtc accatcagga caagccatta accgagcgac cgaaggttcc aatactagtg tggatttacg gcgggggtta catgagtggc acggcgacac ttgatctata taaagccgac ataatggcgt cttcgagtga tgtgatcgta gcctcgatgc agtatagggt tggcgcgttt ggattttttttttt
- the sequence is Plutella xylostella acetylcholinesterase 1 mRNA, GenBank: AY970293.1 nucleotide #s: 889-1188; tc acaatgtcac attgtttgga gaatcgtccg gtgcagtttc cgtgtcatta cacttactgt ctccgctgtc aagaaacatg tttctcaag ctattatgca atctgcagcc gcatctgcac cttgggccat catttccaga gaggagagtg tgataagggg catccgcctg gccgaggccg tccactgc (SEQ ID NO: 2).
- the sequence is Plutella xylostella tyrosine hydroxylase mRNA, GenBank: JN410829.1 nucleotide #s: 301-600; gctgaggtcg gtggaataga cggaaatgca gatgatgatt acaccttgac cgaggaggag gtgatcttgc agaactccgc cagcgagtcc ccggaggccg agcaggcgct gcaacaagcg gctttgctgcgcgacggcatg ggctcgctcg cgcgcgacggcatg ggctcgctcg cgcgcgcatcct caagaccatc gacaactaca agggatgcgt tcaacacctc gagactcgcc ctccaacgc caacg
- the sequence is Plutella xylostella integrin beta 1 mRNA, GenBank: GQ178290.1 nucleotide #s: 531-830; tgcaggtcaa gccgcagagg gtcaagctgc agctgcgcat gaaccagatg cagaaactag acgtcgccta ttcccaagcc caagactacc cggtggacct gtactacttg atggacctga gtcgttccat gaagaacgac aaaggagaagc tcagtacatt gggcagtctg ctgtccagca ctatgaggaa tatcacgcccc aacttccgtc ttggggcttcgg ctttcgtgtgtgtgtgtccagca ctatgaggaa
- the sequence is Plutella xylostella charged multivesicular body protein 4b-like mRNA (XM_011555904.1) nucleotide #s: 321-620; ccaggaaaca tggcactaag aacaaaagag cggccatcgc tgcacttaaa cgcaagaagc gttacgagaa gcaactcaca cagattgacg gcacgctcag ccagatagag atgcagagag aggcattgga gggcgccaac actaacactc aagtactgaa cacgatgcga gaggccgcg cggctatgaa gctctcac aaggatattg acgtagacaa agtgcatgat atcatggacg acatcgctga acaacatgat atcatggacg acatcg
- the sequence is Plutella xylostella peptidoglycan recognition protein mRNA, GenBank: EU399240.1 nucleotide #s: 31-330; tcagtgt tttgttgttg tgctcatgca gggtgtggcg tggtgaccag acagcagtgg gatgggctgg acccgataca gttggagtac ctgcccggccggcccccctggggct ggtggtggtc cagcacaccg ccaccccgc gtgtgacact gacgccgcgt gtgtggagct ggtgcagaa atacagacca atcatatgga tgtgctgaag tttgggata ttggaccgaa cttcctgatt ggt (SEQ ID
- the sequence is Plutella xylostella mRNA for vacuolar ATP synthethase subunit E, GenBank: AB189032.1 (identical to NM_001305532.1) nucleotide #s: 64-363; gcgctca gcgatgcaga tgtccaaaa cagatcaagc atatgatggc cttcatcgag caagaggcaa atgaaaaggc cgaagaaatc gatgctaagg ctgaggagga gttcaacatc gagaaggggc gtctggtgca gcagcagcgc ctcaagatca tggagtacta cgagaagaag gagaagcagg tggaactcca gaagaagatc caatcctcca acatgctgaa ccaggcccgt ctgaaggtg
- the sequence is Diabrotica virgifera virgifera charged multivesicular body protein 4b (LOCI 14337301), mRNA Sequence ID:
- the sequence is Acyrthosiphon pisum V-type proton ATPase subunit E Genbank#: XM_008185078.2 nucleotide#s: 540-724; t tagccaacac tggaataaac gtcaaaataa acattgataa aagtattaaa ttaccgactc aagaaatagg aggcgtcgtg gtcacgtcca aagatcgaag ggtacatgtt gaaaatacgc ttgtagtgag attgctctat cacccaac aaagcaatacc aataatatgc actggactgt ttgg (SEQ ID NO: 9).
- the sequence is Solenopsis invi eta’s (RIF A) actin muscle (LOC105205816, GenBank: XM_011175337.1 nucleotides# 465-763; gatctc tctccctcga ctctaacacc agegaaagta acagccaatc aagatgtgtg aegatgatgt tgcggcatta gtcgtggaca atgggtccgg tatgtgeaag getggatteg cgggggatga tgcaccacgc gctgtgttttc ccagcatcgt cggtcgtcct cgtcatcagg gtgtgatggt cggtatgggt caaaaagaca gttatgttgg egaegaggeg caaagtaaga gaggtatatt gacact
- the sequence is Gibberella zeae isolate NX3 cytochrome P450 5 IB gene GenBank: FJ216402.1 nucleotide positions 804-1023; cagcaag tttgaegagt ccctggccgc tctctaccac gacctcgata tgggcttcac ccccatcaac ttcatgcttc actgggcccc tccctgg aaccgtaagc gcgaccacgc ccagcgcact gttgccaaga tctacatgga cactatcaag gagcgccgcg ccaagggcaa caacgaatcc gagcatgaca tgatgaagca ccttatgaac tet (SEQ ID NO: H).
- the sequence is Fusarium graminearum PH-1 cytochrome P450 51 NCBI Reference Sequence: XM 011327038.1 nucleotide positions 163-400; attggaag caccgtacaa tatggcatcg acccgtacgc ttttttcttc gaetgeagag ataaataegg egaetgettt acctttattc tccttggcaa atcaacgact gtctttcttg gtcccaaggg caatgacttt atcctcaacg gcaaacacgc cgatctcaac gccgaggacg tttatgggaa acttaccacg cccgtgtttg gtgaggaggtttatgac tgctccaatg (SEQ
- MdsRNA post-transcriptionally chemically modified double strand RNA
- the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
- B is a nucleobase
- R 1 is selected from: wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12- 1000, b is an integer from 12-1000, and c is an integer from 12-1000; and the method comprising:
- MdsRNA post-transcriptionally chemically modified double strand RNA
- the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
- B is a nucleobase
- R 1 is selected from: wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12- 1000, b is an integer from 12-1000, and c is an integer from 12-1000; and optionally wherein at least about 2% of all the nucleotides independently comprise
- B is a nucleobase
- R 2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5-C12 heteroaryl, wherein R 2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or C1-C12 alkoxy; the method comprising:
- (a) and (b) are carried out in an anhydrous solvent.
- (c) and (d) are carried out in an anhydrous solvent.
- the anhydrous solvent is selected from DMSO or DCM.
- an ionic solvent is added after (a).
- an ionic solvent is added after (c).
- the ionic solvent is selected from benzyltributyl ammonium chloride or benzyltrimethyl ammonium chloride.
- the activation agent is carbonyldiimidazole.
- the suitable leaving group is: wherein X / ' represents the covalent point of attachment to carbonyl of Formula (IIA).
- the suitable leaving group is: wherein »AAA/' represents the covalent point of attachment to carbonyl of Formula (VA).
- (b) has a ratio of less than ten equivalents of the compound of Formula (IIA) per nucleotide of the dsRNA.
- (d) has a ratio of between about two equivalents and about fifty equivalents of the compound of Formula (VA) per nucleotide of the dsRNA.
- (b) has a ratio of less than two equivalents of the compound of Formula (IIA) per nucleotide of the dsRNA.
- (d) has a ratio of between about four equivalents and about twenty -five equivalents of the compound of Formula (VA) per nucleotide of the dsRNA.
- the compound of Formula (II) is the anhydride.
- the compound of Formula (V) is the anhydride.
- R 1 is selected from:
- x is an integer from 80-1000.
- x is an integer from 50-900.
- a is an integer from 180-1000.
- b is an integer from 80-1000.
- c is an integer from 80-1000.
- R 1 has a molecular weight between 5,000 and 10,000 Da.
- R 1 has a molecular weight between 5,000 and 40,000 Da.
- R 1 is polyethylene glycol (PEG).
- R 1 is poloxamer 407, 338, 188, 184, 401, or any combination thereof.
- a is 101
- b is 56
- c is 101
- R 1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R 1 is a HMW polymer with an HLB ranging from about 8 to about 27.
- steps (c) and (d) are present.
- steps (b) and (d) are performed sequentially.
- steps (b) and (d) are performed simultaneously.
- the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect, fungus, weed or acari, as discussed in detail in this disclosure, such as but not limited to target (insect fungus, weed or acari), target sequences and target regions.
- R 2 is selected from N-methyl anthranoyl (NMA), N- benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- NMA N-methyl anthranoyl
- NBA N-benzyl anthranoyl
- dimethyl furoyl -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- compositions methods of making the compositions, nucleotide sequences and uses for target insects, fungi, weeds or acari are intended to be covered herein this disclosure.
- the disclosed method of preparation is superior to prior methods in both cost and scalability.
- Prior methods of synthesis required water or another suitable polar protic solvent to reduce the need for excess solvent dilution. However, this led to significant degradation of the necessary polyalkyloxy polymer anhydrides.
- Prior methods sometimes employed as much as 200 equivalents of modifying groups per one bp of dsRNA.
- the addition of ionic solvent has provided the superior and unexpected benefit of reducing the amount of solvent used, reducing the degradation of starting material, and allowing efficient synthesis of the desired product.
- (b) has a ratio of less than 0.05 equivalents of the compound of Formula (IIA) (e.g., the polyalkyloxy polymer) per one bp of the dsRNA. In another embodiment of the method, (b) has a ratio of less than 0.1 equivalents of the compound of Formula (IIA). In particular embodiments of the method, (b) has a ratio of about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 equivalents of the compound of Formula (IIA) per one bp of the dsRNA.
- the ionic solvent is not acting as a phase transfer catalyst. Rather, the ionic solvent is acting, unexpectedly, as a superior solvating agent.
- the ionic solvent also serves to help “unzip” the dsRNA by shielding the charges on the molecules. This use of such an ionic solvent is unreported in the prior art.
- the ionic solvent improves dsRNA dissociation by shielding the resulting charge on the single strand RNA.
- MdsRNA post-transcriptionally modified double strand RNA
- the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (VI): or an acceptable salt thereof, wherein:
- B is a nucleobase
- R 3 is selected from: amino acids, fatty acids, alkyl; substituted alkyl; alkenyl; substituted alkenyl; alkynyl; substituted alkynyl; aryl; substituted aryl; Cl -CIO alkyl, Cl- C10 alkenyl, or Cl -CIO alkynyl wherein alkyl and alkenyl can be linear, branched or cyclic; hydrogen; methyl; ethyl; propyl; isopropyl; butyl; isobutyl; tert-butyl; pentyl; hexyl; cyclohexyl; heptyl; octyl; nonyl; decyl; vinyl; allyl; ethynyl; benzyl; cinnamyl; COCM aryl; C6-C14 substituted aryl; heterocyclyl; C5-C14 heterocyclyl; phenyl; mono
- (a) and (b) are carried out in an anhydrous solvent.
- the anhydrous solvent is selected from DMSO or DCM.
- an ionic solvent is added after (a).
- the ionic solvent is selected from benzyltributyl ammonium chloride or benzyltrimethyl ammonium chloride.
- the activation agent is carbonyldiimidazole.
- the suitable leaving group is: wherein »AAA/' represents the covalent point of attachment to carbonyl of Formula (IVA).
- (b) has a ratio of between about two equivalents and about fifty equivalents of the compound of Formula (IVA) per nucleotide of the dsRNA.
- (b) has a ratio of between about four equivalents and about twenty -five equivalents of the compound of Formula (IVA) per nucleotide of the dsRNA.
- the compound of Formula (IV) is the anhydride.
- R 3 is selected from N-methyl anthranoyl (NMA), N- benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- NMA N-methyl anthranoyl
- NBA N-benzyl anthranoyl
- dimethyl furoyl -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
- the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect, fungus, weed or acari, as discussed in detail in this disclosure, such as but not limited to target (insect fungus, weed or acari), target sequences and target regions.
- compositions methods of making the compositions, nucleotide sequences and uses for target insects, fungi, weeds or acari are intended to be covered in this disclosure.
- the disclosed method of preparation is superior to prior methods in both cost and scalability.
- Prior methods of synthesis required water or another suitable polar protic solvent to reduce the need for excess solvent dilution. However, this led to significant degradation of the necessary anhydrides.
- Prior methods sometimes employed as much as 200 equivalents of modifying groups per one bp of dsRNA.
- the addition of ionic solvent has provided the superior and unexpected benefit of reducing the amount of solvent used, reducing the degradation of starting material, and allowing efficient synthesis of the desired product.
- (b) has a ratio of less than 0.05 equivalents of the compound of Formula (IVA) per one bp of the dsRNA.
- (b) has a ration of less than 0.1 equivalents of the compound of Formula (IVA). In particular embodiments of the method, (b) has a ratio of 0.2 equivalents of the compound of Formula (IVA) per one bp of the dsRNA.
- the ionic solvent is not acting as a phase transfer catalyst. Rather, the ionic solvent is acting, unexpectedly, as a superior solvating agent.
- the ionic solvent also serves to help “unzip” the dsRNA by shielding the charges on the molecules. This use of such an ionic solvent is unreported in the prior art.
- a method of modifying the expression of a polynucleotide of interest in an insect comprising administering a composition of the disclosure.
- a target gene is selected such that inhibiting expression of the target gene kills, inhibits growth or appetite of, or slows reproduction of an animal, fungus, or weed. Inhibiting expression of the target gene can control, kill, inhibit growth or appetite of, or slow reproduction of the animal, fungus, or weed.
- the insect, fungus, or plant is of agricultural significance.
- an agriculturally significant animal, fungus, or plant is an insect, fungus, or weed.
- the modified expression reduces the fertility rate of the target insect.
- the described MdsRNAs can be used to control, kill, inhibit growth, appetite, or feeding of, or slow reproduction of an animal, fungus, or plant in an agricultural or urban setting.
- a plant target gene is selected such that inhibiting expression of the gene in the plant increases plant growth, viability, quality, or yield.
- the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect.
- an efficacious sequence or target gene useful in the products and methods of the disclosure will inhibit the expression of the target gene, act on the mid gut of the insect and increase mortality or induce growth stunting, or stop instar development.
- the target insect is Diamondback moth (Plutella xylostella), Gypsy moth, Red imported fire ant (Solenopsis invicta), Fall armyworm, Colorado potato beetle, Canola flea beetle, Aedes aegypti, or Western corn root worm (Diabrotica virgifera virgifera).
- the target insect is Pea aphid (Acyrthosiphon pisum), Soybean aphid, Piezodorus guildinii.
- the target is an acari, such as Verroa mite.
- the target is a weed, such as Palmer amaranth.
- the target is a fungus such as Palmer amaranth, Fusarium graminearum (Gibberella zeae) or Botrytis.
- the MdsRNA used in the compositions of the disclosure will be a nucleotide sequence that can inhibit the expression of target genes or regions in these insects.
- the MdsRNA comprises a sequence complementary to a target region in Diamondback moth, such as but not limited to AChE2, P450, CYP6BFlvl, DOUX, Cytokine receptor DOMELESS, Protein MESH transcript variant XI, Venom carboylesterase-6 and VPASE-E.
- the MdsRNA comprises a sequence complementary to a target region in Fall armyworm, such as but not limited to P450, Cytokine receptor DOMELESS, VPASE, Dredd, Protein MESH transcript variant XI, P450 CYP9A58, P450 CYP321 A8, P450 CYP6B2-like.
- a target region in Fall armyworm such as but not limited to P450, Cytokine receptor DOMELESS, VPASE, Dredd, Protein MESH transcript variant XI, P450 CYP9A58, P450 CYP321 A8, P450 CYP6B2-like.
- the MdsRNA comprises a sequence complementary to a target region in Western corn root worm, such as but not limited to SNF7.
- the MdsRNA comprises a sequence selected from one of SEQ ID Nos. 1-269 and its perfect or imperfect reverse complementary strand.
- the sequence is selected from one of SEQ ID Nos. 13 or 248-251 and its perfect or imperfect reverse complementary strand.
- the target insect is a Lepidopteran and can be targeted using any one of SEQ ID Nos. 1-269 and its perfect or imperfect reverse complementary strand thereof for each sequence specified.
- the Lepidopteran is targeted using any one of SEQ ID Nos. 13 or 248- 251.
- the target insect is a Lepidopteran.
- the Lepidopteran is an army worm, com ear worm, cabbage butterfly, or cotton boll worm.
- the MdsRNA comprises a sequence complementary to the P450, CYP6FBlvl, MESH, AChE2, VPASE, DOMELESS, DOUX or Venom target region in a Lepidopteran.
- the treatment of at least about 30% control of the target insect In an embodiment, the treatment of at least about 50% control of the target insect. In an embodiment, the treatment shows greater than about 50% control of the target insect. In an embodiment, the treatment shows between about 50% to about 90% control of the target insect. In an embodiment, the treatment shows between about 50% to about 75% control of the target insect. In an embodiment, the treatment shows between about 30% to about 50% control of the target insect.
- compositions and methods herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present disclosure. Theoretical aspects are presented with the understanding that Applicant does not seek to be bound by the theory presented. All parts or amounts, unless otherwise specified, are by weight.
- compositions containing the described MdsRNAs are described.
- the MdsRNA-containing compositions are formulated for agricultural application (agrochemical compositions).
- an agrochemical composition comprises an effective amount of at least one MdsRNA and optionally one or more acceptable carriers or excipients.
- Carriers and excipients are substances other than the MdsRNA that have been appropriately evaluated for safety and are intentionally included in a composition. Excipients may act to a) aid in processing of the MdsRNA during manufacture, b) protect, support or enhance stability or bioavailability of the MdsRNA, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the MdsRNA during storage or use.
- An acceptable carrier or excipient may or may not be an inert substance.
- "effective amount,” refers to that amount of a MdsRNA to produce the intended result.
- Carrier and excipients include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents (pH regulating agents), chelating agents, coating agents, colors, delivery enhancers, dextran, dextrose, diluents, disintegrates, dispersants, emulsifiers, extenders, fillers, foam control agents, glidants, humectants, lubricants, oils, pigments, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
- absorption enhancers include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents (pH regulating agents),
- an agrochemical composition comprises one or more adjuvants or surfactants.
- the one or more adjuvant or surfactants are independently selected from anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, anti-condensates, thickeners, emulsifiers, spreaders, stickers, organosilanes, fatty esters and oils.
- the one or more adjuvants or surfactants are optionally selected from non-ionic, organo silicone surfactants (e.g., KINETIC ® from the Helena company; nonionic organosilicone-based wetter/spreader/penetrant spray adjuvant), DYNE-AMIC ® (from the Helena company; blend of highly refined methylated seed oils in combination with specialized organosilicone-based nonionic surfactants) and SIL WET ® (from Momentive; nonionic surfactant).
- non-ionic, organo silicone surfactants e.g., KINETIC ® from the Helena company; nonionic organosilicone-based wetter/spreader/penetrant spray adjuvant
- DYNE-AMIC ® from the Helena company; blend of highly refined methylated seed oils in combination with specialized organosilicone-based nonionic surfactants
- SIL WET ® from Momentive; nonionic surfactant
- an agrochemical composition comprises one or more agents selected from an herbicide, fungicide, insecticide, acaricide and fertilizer.
- the described MdsRNAs and compositions containing MdsRNAs can be processed in a number of different ways known to those skilled in the art to facilitate application of such material onto plants or into baits and for use in the field or in urban environments.
- the described MdsRNAs and compositions comprising MdsRNAs disclosed herein can be packaged or included in a kit, container, pack, or dispenser.
- an agrochemical composition contains two or more different MdsRNAs.
- the MdsRNAs may have different antisense sequences complementary to the same target gene, different antisense sequences complementary to different crizt genes in the same or different hosts, different or similar lengths, or different or similar post transcriptional modification.
- an agrochemical composition is an emulsifiable agricultural concentrate.
- an emulsifiable agricultural concentrate further contains a least one agent that can be, but is not limited to, carrier, or organic solvent, surfactant, excipient, herbicide, fungicide, insecticide, fertilizer, or combinations thereof.
- an agrochemical composition contains one or more herbicides.
- suitable herbicides include, but are not limited to, imidazolinone, acetochlor, acifluorfen, aclonifen, acrolein, AKH-7088, alachlor, alloxydim, ametryn, amidosulfuron, amitrole, ammonium sulfamate, anilofos, asulam, atrazine, azafenidin, azimsulfuron, BAS 620H, BAS 654 OOH, BAY FOE 5043, benazolin, benfluralin, benfuresate, bensulfuron-methyl, bensulide, bentazone, benzofenap, bifenox, bilanafos, bispyribac-sodium, bromacil, bromobutide, bromofenoxim, bromoxynil, butachlor
- an agrochemical composition contains one or more fungicides.
- Suitable fungicides include, but are not limited to, carbamate fungicides such as 3,3'-ethylenebis(tetrahydro-4,6-dimethyl-2H-l,3,5-thiadiazine-2-thione), zinc or manganese ethylenebis(dithiocarbamate), bis(dimethyldithiocarbamoyl)disulfide, zinc propylene-bis-(dithiocarbamate), bis(dimethyldithiocarbamoyl)ethylenediamine, nickel dimethyl-dithiocarbamate, methyl 1 -(butylcarbamoyl)-2-benzimidazolecarbamate, l,2-bis(3-methoxycarbonyl-2-thioureido)benzene, l-isopropylcarbamoyl-3-(3,5- dichlorophenyl)-hy
- an agrochemical composition contains one or more insecticides.
- suitable insecticides include, but are not limited to, phosphoric insecticides such as 0,0-diethyl O-(2-isopropyl-4-methyl-6-pyrimidinyl)phosphorothioate, 0,0-dimethyl S-2-[(ethylthio)ethyl]phosphorodithioate, 0,0-dimethyl O-(3-methyl-4- nitrophenyl)-thiophosphate, 0,0-dimethyl S-(N-methylcarbamoylmethyl)- phosphorodithioate, 0,0-dimethyl S-(N-methyl-N-formylcarbamoylmethyl) phosphoro- dithioate, O,O-dimethyl S-2-[(ethylthio)ethyl] phosphorodithioate, 0,0-diethyl S-2-[(ethylthio)ethyl] phosphorodithioate,
- an agrochemical composition contains one or more fertilizers.
- a variety of fertilizers are suitable for inclusion in the compositions.
- the fertilizer can be a single nutrient fertilizer (N, P, or K), binary fertilizer (e.g., NP, NK, or PK), a NPK fertilizer, or a multinutrient fertilizer (e.g., may provide one or more of calcium, magnesium, sulfur, copper, iron, manganese, molybdenum, zinc, boron, silicon, cobalt, or vanadium).
- the fertilizer can be of natural origin or synthetic origin.
- the fertilizer can be liquid or solid, and may provide slow or controlled release.
- the MdsRNAs comprise less than 50% by weight of a composition.
- the amount of MdsRNA in an agriculture composition is less than 5% by weight of the composition.
- the MdsRNA is present in the composition in an amount less than about 1% by weight, less than about 0.9% by weight, less than about 0.8% by weight, less than about 0.7% by weight, less than about 0.6% by weight, less than about 0.5% by weight, less than about 0.4% by weight, less than about 0.3% by weight, less than about 0.2% by weight, less than about 0.1% by weight, less than about 0.05% by weight, less than about 0.01% by weight, or less than about 0.001% by weight of the composition.
- the agrochemical composition is formulated as a liquid.
- Liquid formulations can be prepared by mixing the MdsRNA and other agents in a liquid until dissolution of all the components is achieved in the weight percentages described below.
- the liquid can be an aqueous, ionic, or organic liquid.
- Suitable liquids include, but are not limited to, water, alcohols (e.g. methanol and ethanol), ketones (e.g. acetone, methyl ethyl ketone and cyclohexanone), aromatic hydrocarbons (e.g.
- benzene, toluene, xylene, ethylbenzene and methylnaphthalene aliphatic hydrocarbons (e.g. hexane and kerosene), esters (e.g. ethyl acetate and butyl acetate), nitriles (e.g. acetonitrile and isobutyronitrile), ethers (e.g. dioxane and diisopropyl ether), acid amides (e.g. dimethylformamide and dimethylacetamide), and halogenated hydrocarbons (e.g. di chloroethane, trichloroethylene and carbon tetrachloride).
- aliphatic hydrocarbons e.g. hexane and kerosene
- esters e.g. ethyl acetate and butyl acetate
- nitriles e.g. acetonitrile and isobut
- the liquid formulation is an aqueous formulation.
- an aqueous formulation contains only water, the MdsRNA and other agents.
- additional compounds, solvents, or adjuvants are provided with the aqueous formulation.
- the agrochemical composition is formulated as a powder or dust.
- the powder or dust can be granulated to be suitable for applying the powder or dust directly to a crop (i.e., by dusting the crop), or it can be granulated for eventual dissolution in a solvent such as water.
- the composition is a lyophilisate.
- the MdsRNA and the other agents are lyophilized together.
- one or more MdsRNAs and the other agents can be lyophilized separately.
- Suitable solid and gaseous carriers can be utilized in the compositions.
- Suitable solid carriers include, but are not limited to, fine powders or granules of clays (e.g. kaolin clay, diatomaceous earth, synthetic hydrated silicon dioxide, attapulgite clay, bentonite and acid clay), talcs, bulking agents, inorganic minerals (e.g., sericite, powdered quartz, powdered sulfur, activated carbon, calcium carbonate and hydrated silica), and salts for chemical fertilizers (e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea and ammonium chloride).
- Suitable gaseous carriers include, for example, butane gas, carbon dioxide, and fluorocarbon gas.
- an agrochemical composition includes a dispersant.
- dispersants include, but are not limited to, methyl cellulose, polyvinyl alcohol, sodium lignin sulfonates, polymeric alkyl naphthalene sulfonates, sodium naphthalene sulfonate, polymethylene bisnaphthalene sulfonate, neutralized polyoxyethylated derivatives, and ring-substituted alkyl phenol phosphates.
- Stabilizers may also be used to produce stable emulsions.
- Exemplary stabilizers include, but are not limited to magnesium, aluminum silicate, and xanthan gum.
- an agrochemical composition is formulated as a spray in the form of an aerosol.
- the composition When formulated as an aerosol spray, the composition is generally charged in a container under pressure together with a propellant.
- propellants include fluorotrichloromethane and dichlorodifluoromethane.
- an agrochemical composition includes a seed.
- an agrochemical composition comprises an antifungal MdsRNA and a seed.
- an agrochemical composition comprises a MdsRNA, a seed, and further comprises a fungicide.
- the amount of the MdsRNA in a fungicidal composition is less than about 5% by weight, less than about 1% by weight, less than about 0.9% by weight, less than about 0.8% by weight, less than about 0.7% by weight, less than about 0.6% by weight, less than about 0.5% by weight, less than about 0.4% by weight, less than about 0.3% by weight, less than about 0.2% by weight, less than about 0.1% by weight, less than about 0.05% by weight, less than about 0.01% by weight, or less than about 0.001% by weight of the fungicidal composition.
- the weight of the fungicidal composition does not include the weight of the seed.
- the fungicidal composition is present inside the seed coat, or internal to the seed. In some embodiments, the fungicidal composition is formed over the seed such that it covers the exterior of the seed, either fully or partially. Methods for coating a seed include those known in the art.
- MdsRNAs or compositions containing MdsRNAs are used to control agricultural pests or treat agricultural pest infestation.
- the MdsRNAs can be administered to the pest, to an area occupied by the pest, or to a food source of the pest.
- methods are provided for treating for or controlling pests.
- the pest is an insect, fungus, acari or weed.
- the methods comprise applying a composition comprising one or more described MdsRNAs to an area to be treated.
- the MdsRNA is present in the composition in an amount of less than 5% by weight.
- the composition is applied directly to a surface.
- the surface is a plant surface upon which the targeted insect or fungal pest feeds.
- the gene expression level and/or mRNA level of a target gene in a target host is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, following application of MdsRNAs or MdsRNA-containing composition.
- mortality of the agricultural pest in increased at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% following application of MdsRNAs or MdsRNA-containing composition.
- controlling a pest means to reduce crop damage or decreased yield caused by the pest, or to increase morbidity, inhibit growth or appetite or feeding of, or slow reproduction of the pest compared with the damage, decreased yield morbidity, growth, appetite, feeding, or reproduction as measured in the absence of treatment with MdsRNAs.
- methods of reducing expression of a target gene in a target plant other than a weed comprise applying a composition containing one or more of the described MdsRNAs to the plant.
- the plant is a crop plant.
- a crop plant is a plant that can be grown and harvested for profit or subsistence.
- a crop plant can be, but is not limited to, a food plant, horticultural plant, Horticulture plant, or industrial plant.
- the plant is a cultivated plant. The plant can be in a laboratory, greenhouse, nursery, field, orchard or other agricultural setting, garden, or another natural or urban setting.
- a target plant is a plant considered desirable in a particular situation or location.
- the animal is an insect.
- the insect is a Coleopteran (such as a beetle).
- a Coleopteran can be, but are not limited to, bark beetle, elm leaf beetle, Asian longhorn beetle, death watch beetle, mountain pine beetle, coconut hispine beetle or the Colorado potato beetle.
- the insect is a Lepidopteran (such as a butterfly or moth).
- a Lepidopteran can be, but is not limited to, army worm, corn ear worm, cabbage butterfly, or cotton boll worm.
- the insect is a Hymenopteran (such as sawflies, wasps, bees, ants).
- a Hymenopteran can be, but is not limited to, fire ant, argentine ant, carpenter ant, leafcutter ant, army ant, wheat stem sawfly, larch sawfly, spruce sawfly, or bed bug.
- the insect is a Dipteran (such as a fly).
- a Dipteran can be, but is limited to, fly, mosquito, gnat, or leafminer.
- the insect is a Hemipteran (such as a true bug).
- a Hemipteran can be, but is not limited to, aphid, hopper, bug, whiteflies, mealybug, or flea.
- the insect is a Western corn root worm.
- the insect is an insect having resistance to one or more conventional known insecticides.
- the insect such as a Red imported fire ant has the potential to have a negative impact on biodiversity (Wojcik et al. 2001 and/or resistance to insecticides (Zhang et al. 2016).
- the insect such as a mosquito, has the potential to impact human health as a vector for disease, such as, but not limited to, Malaria, Dengue, Zika and Chikungunya (Hemingway et al. 2004).
- the insect such as Asian citrus psyllid, is a vector of the citrus greening disease (Tiwari et al. 2011).
- RNAi RNA-derived neuropeptides
- Field application of naked RNAs is generally impractical due to the sensitivity of RNA to environmental specific and nonspecific degradation (Baum 2016).
- RNA is highly susceptible to degradation during the course of feeding and in transit through the insect gut.
- Lepidoptera seem to degrade RNA much more aggressively than the Coleoptera, which may account for their relatively poor susceptibility to RNAi mediated control methods.
- the stability of the described MdsRNAs serves to protect the MdsRNA from host nucleases before delivery to the RNAi pathway, and limits nonspecific environmental degradation.
- the described MdsRNAs are nevertheless sufficiently biodegradable to be considered environmentally safe.
- a composition comprising one or more MdsRNAs can be applied to a plant prior to infection to prevent an insect infection.
- the composition may also be applied after the appearance of signs of infection to treat an insect infection.
- the composition can be applied by a variety of methods depending on the plant part to be treated.
- the composition can be applied to a plant seed prior to planting to prevent insect infection of the seed.
- the composition can be applied to the soil at the time of planting or just before planting to prevent insect infestation of the newly planted seed (i.e., as a pre- emergent).
- the composition can be applied to a plant after its germination or to the foliage of the plant after emergence to either treat or prevent insect infestation (i.e., as a post-emergent).
- the application occurs during the stages of germination, seedling growth, vegetative growth, and reproductive growth. In some embodiments, application occurs during vegetative and reproductive growth stages.
- Applying the composition to a pre-emergent seed may involve various seed coating techniques such as film coating, pelleting, encapsulation, drum coating, and fluidized bed coating. Applying to a post-emergent plant may involve spraying or crop dusting techniques.
- an effective amount of the composition can be applied to a plant or seed by several methods generally known in the art.
- the amount of composition comprising an “an effective amount” can and will vary depending upon the plant and its stage of production, the fungal target, and environmental conditions.
- the plant or its progeny is treated with an amount of the composition sufficient to provide a concentration of active ingredients from about 0.01 mg/kg to about 10% by weight.
- the method may involve more than one application of the composition to the plant or its progeny.
- the number of applications may range from about 1 to about 5 or more.
- the applications, as detailed herein, can be applied at the same or different stages of the plant’s life cycle.
- the MdsRNAs are used to treat or prevent fungal infection.
- the fungus can be, but is not limited to, a Hypocrealesan, Venturia, Podosphaera, Erysiphe, Monolinia, Mycosphaerella, Uncinula; Basidiomycete, Hemileia, Rhizoctonia, Puccinia, Fungi imperfecti, Botrytis, Helminthosporium, Rhynchosporium, Fusarium, Septoria, Cercospora, Alternaria, Pyricularia, Pseudocercosporella, Oomycete fungi, Phytophthora, Peronospora, Bremia, Pythium, Plasmopara, Phakopsora Pachyrhizi, P.
- treatment of Fusarium graminearum can reduce the production of mycotoxins, the risk of emergence of resistance to fungicides based on demethylation inhibitors (DMI), orcarcinogenicity concerns about conventional DMI like Tebuconazole.
- DMI demethylation inhibitors
- the described agrochemical compositions can be applied to a plant prior to infection to prevent a fungal infection.
- the described agrochemical compositions can be applied to a plant after the appearance of signs of infection to treat a fungal infection.
- the composition can be applied by a variety of methods depending on the plant part to be treated.
- the composition can be applied to a plant seed prior to planting to prevent fungal infection of the seed.
- the composition can be applied to the soil at the time of planting or just before planting to prevent microbial infestation of the newly planted seed (i.e., as a pre-emergent).
- the composition can be applied to a plant after its germination or to the foliage of the plant after emergence to either treat or prevent microbial infestation (i.e., as a post emergent).
- the application occurs during the stages of germination, seedling growth, vegetative growth, and reproductive growth. More typically, applications occur during vegetative and reproductive growth stages.
- Applying the composition to a pre-emergent seed may involve various seed coating techniques such as film coating, pelleting, encapsulation, drum coating, and fluidized bed coating. Applying to a post-emergent plant may involve spraying or crop dusting techniques.
- an effective amount of the composition can be applied to a plant or seed by several methods generally known in the art.
- the amount of composition comprising an “an effective amount” can and will vary depending upon the plant and its stage of production, the fungal target, and environmental conditions.
- the plant or its progeny is treated with an amount of the composition sufficient to provide a concentration of active ingredients from about 0.01 mg/kg to about 5,000 mg/kg.
- the method may involve more than one application of the composition to the plant or its progeny.
- the number of applications may range from about 1 to about 5 or more.
- the applications, as detailed herein, can be made at the same or different stages of the plant’s life cycle.
- a weed is a plant considered undesirable in a particular situation or location.
- a weed can be, but is not limited to, Palmer Amaranth, Common Lambsquarters, Horseweed, Morning Glory, Waterhemp, Nutsedge, Kochia, Common Ragweed, Giant Ragweed, or Nightshade.
- Example 1 Preparation of High Molecular Weight Polyalkyloxy Polymer dsRNA in DMSO.
- Benzyl triethyl ammonium chloride (Benzyl-TriBA-Cl) was dissolved in DMSO at 200 mM. An aliquot of this solution was added to freeze-dried dsRNA in a reaction tube. The tube was warmed to 65°C in a shaker for 1 hr. Then the temperature was reduced to 55°C and catalytic amount of pyridine was added followed by a 200 pg/pL solution of DMAP. A 200 ug/uL solution of activated PEG was added in 1-3 portions.
- the mixture was kept at 55 °C for 90 min and then was quenched with 500 mM citrate pH 4.5, and diluted to 40 mL with water.
- the resulting mixture containing the crude PEG- dsRNA was purified using tangential flow filtration, TFF, to yield MdsRNA with >90% purity.
- Table 1 Modified MdsRNA Compounds Synthesized by Procedure of Example 1.
- a Input is moles of PEG/mole of RNA nucleotide.
- b Modifier is the -OC(O)R 1 modification as defined in Formula I.
- c Sequence is the sequence of nucelobases of the RNA strand backbone.
- the agarose gel (2%) analysis of the activated 10k PEG/ dsRNA reaction mixture (line 3) demonstrates the covalent modification of the 300 bp molecule with 1, 2, 3, 4 and so on chains of 10k PEG polymer. The original unmodified dsRNA molecule is all consumed during the reaction.
- Example 2 Preparation of High Molecular Weight Polyalkyloxy Polymer dsRNA using DCM for the activation reaction.
- the viscous liquid was triturated with 2 volumes of acetonitrile (CAN) and stripped under a stream of argon to remover water and then dried under high vac and P2O5 for several hours.
- the resulting solid was reacted with dsRNA following the ionic solvation method.
- the following MdsRNAs were prepared according to this procedure:
- Table 2 Modified MdsRNA Compounds Synthesized by Procedure of Example 2.
- a Input is moles of PEG/mole of RNA nucleotide.
- b Modifier is the -OC(O)R 1 modification as defined in Formula I.
- c Sequence is the sequence of nucelobases of the RNA strand backbone.
- Example 3 Preparation of High Molecular Weight Polyalkyloxy Polymer dsRNA in DMSO.
- the polyaloxy polymer was reacted with a linker to introduce a reactive COOH at one end of the polymer chain.
- One eq. of polyalkyloxy polymer was dissolved in 1,4- dioxane.
- DMAP 0.5 eq.
- DIEA 3 eq.
- the corresponding anhydride 1.5. eq.
- the reaction mixture was stirred overnight at room temperature.
- the mixture was diluted with DCM and extracted with sodium bicarbonate, brine.
- the organic layer was dried over sodium sulfate, and concentrated at reduced pressure.
- the resulting gum was triturated with fresh DCM and the solid polyalkyloxy-COOH was collected via filtration.
- Methoxy PEG acetic acid IK, 2K, 5K, and 10K
- Methoxy PEG acetic acid MPEG A- Y 40K, Y-shaped
- Methoxy PEG succinic acid MPEGS
- Methoxy PEG glutamic acid MPEGG
- Methoxy PEG 3, 3 -methylglutamic acid MPEGM
- Carbamoyl PEG 5K CPEG
- Poloxalene succinic L68 Poloxalene succinic L121
- Poloxalene succinic Fl 27, 12.5K Methoxy PEG acetic acid
- MPEG A- Y Methoxy PEG acetic acid
- MPEGM Methoxy PEG 3, 3 -methylglutamic acid
- CPEG Poloxalene succinic L64, 2,9K
- Poloxalene succinic L68 Poloxalene succinic L121
- Benzyl-TriButyl Ammonium-Chloride was dissolved in DMSO at 200 mM. An aliquot of this solution was added to freeze-dried dsRNA in a reaction tube. The tube was warmed to 65°C in a shaker for 1 hr. Then the temperature was reduced to 55°C and catalytic amount of pyridine was added followed by a 200 pg/pL solution of DMAP. A 200 ug/uL solution of activated polyalkyloxy polymer was added in portions. The mixture was kept at 55 °C for 90 min and then was quenched with 500 mM citrate pH 4.5 and diluted to 40 mL with water.
- the resulting mixture containing the crude M-dsRNA was purified using tangential flow filtration (TFF), to yield MdsRNA with >90% purity.
- the MdsRNAs were characterized by gel electrophoresis with non-denaturing agarose gel and denaturing polyacrylamide gel.
- the extent of modification i.e. the ratio of number of bases esterified with polymer to the total number of bases in the dsRNA was determined by a combination hydrolysis/HPLC-ELSD method. Hydrolysis of the MdsRNA samples was obtained upon heating at 99°C a mixture of an aqueous solution of the purified product and 0.5M NaOH solution. The resulting polyalkyloxy polymer and RNA nucleotides were quantified using a Shimadzu LC-2030C HPLC fitted with a Cl 8 100 A LC Column 250x4.6 mm and an ELSD. A calibration curve was built using dsRNA starting material and polyalkyloxy polymer as standards.
- MdsRNAs targeting Diamondback moth (DBM) (P. xylostella) were tested on DBM larvae using cabbage leaf discs.
- DBM Diamondback moth
- the MdsRNA treatments were dissolved in water with an adjuvant and diluted to obtain the desired concentration.
- the desired treatment solution was sprayed on both sides of fresh cabbage leaf discs (3.5 cm diameter). Each treatment was applied with 3 repetitions and a water only control.
- the discs were placed on a wet paper towel in a container.
- P. xylostella eggs (8-12) were transferred to each treated disc and the containers were incubated at 26 °C and 72% relative humidity (RH).
- b Modifier is the -OC(O)R 1 modification as defined in Formula I.
- c Sequence is the sequence of nucleobases of the RNA strand backbone.
- Example 4 Preparation of High Molecular Weight Polyalkyloxy Polymer- NMIA dsRNA.
- MPEGA- dsAChE2 was prepared and purified according to the procedure described in Example 3 using Methoxy PEG acetic acid 10K at input 0.1. Benzyl-TriBA- C1 was dissolved in DMSO at 200 mM. An aliquot (3.4 mL) of this solution was added to freeze-dried MPEGA-dsAChE (34 mg) in a reaction tube. The tube was warmed to 65°C in a shaker for 1 hr.
- Example 5 Preparation of High Molecular Weight Polyalkyloxy Polymer- FA dsRNAs. [00267] Following a similar procedure as described in Example 4. MPEGA-P450 5K (115) was reacted with activated lauryl (LAU), oleic (OLE) and linoleic (LIN) fatty acids to yield the MPEGA-FA-P450 materials described in Table 4.
- LAU lauryl
- OLE oleic
- LIN linoleic
- modified dsRNA materials were tested on DBM larvae using a cabbage leaf disc bioassay and DBM eggs collected from the field.
- Cabbage leaves were collected at stage 4-5. Leaves were prepared by washing with tap water using a nozzle to remove insects, dust etc. The leaves were then wiped with paper towels. Leaf discs of 3.5 cm diameter were punched from the cabbage leaves using a metal cutter. An aqueous solution of the desired treatment solution was prepared at 500 ppm or 150 ppm concentrationand 60uL were sprayed on both sides of the disc. After the treatment was applied to both sides of the leaf disc, four 1 cm diameter discs were then punched from the treated 3.5 cm. The resulting treated 1 cm diameter discs were used in the assay. Three P. xylostella egg wassere placed on each treated disc placed in a petri dish.
- the plates were incubated at 26 °C and 72% room humidity (RH). After 48 hours only one neonate was transferred to a new (2 nd ) treated disc. A third treated disc was provided after 24 hours of the 2 nd treated disc. The larvae were then provided with an untreated disc every day for up to 10 days. Mortality was recorded daily starting on the 2 nd day after incubation. Mortality rate was calculated for each test during the duration of the experiment and cumulative mortality after 5 days and 7 or 9 days was calculated.
- RH room humidity
- Benzyl-TriButyl Ammonium-Chloride is dissolved in DMSO at 200 mM. An aliquot of this solution is added to freeze-dried dsRNA in a reaction tube. The tube is warmed to 65°C in a shaker for 1 hr. Then the temperature is reduced to 55C and 110 uL pyridine are added followed by a 200 ug/uL solution of DMAP and two additions of a 200 ug/uL solution of N-methyl isatoic anhydride (NMIA). The mixture is kept at 55°C for 90 min and then is quenched with 500 mM citrate, and diluted with water.
- NMIA N-methyl isatoic anhydride
- NMA-dsRNA The resulting mixture containing the crude NMA-dsRNA is purified using tangential flow filtration, TFF, to yield MdsRNA with >90% purity.
- the following NMA-dsRNA were prepared according to the procedure described above: NMA-P450(l 15) (37% NMA), NMA- VPASE(7) (81% NMA), NMIA-AChE(l) (40%, 80% NMA), NMA-B1(4), NMA-TH(3). This procedure was also used to prepare dsRNA analogs for NBA, dimethyl furoyl, -Tyr, - Trp, -Leu, and octanoyl. This procedure can be used for other modifications such as, but not limited to, lauroyl, linoleyl, and the like.
- RNAi active ingredient in a field has always been of primary importance in development of advanced pesticides.
- RNA is known to be a very unstable molecule vulnerable to enzymatic degradation.
- Estimations of half-life for dsRNA in the field range between 0.5 and 0.7 days after foliar application on soybean plants (Bachman et al., 2020). The time decay of dsRNA sequences sprayed on cabbage leaves for native dsRNAs and 2’-0 modified dsRNAs was studied in a small plot field trial.
- the dsRNA used in this trial is a dsSNF7 sequence that has been previously reported in field dissipation study (Bachman et al., 2020).
- QuantGene a nucleic acid detection platform marketed by Invitrogen, was the preferred analytical method used to quantify of dsRNA sequences on the cabbage plant leaves after spray.
- the sequence and corresponding Quantigene probe sets were published previously (Armstrong et al., 2013). The sequence is not directed against Diamondback Moth (DBM) and used here only for analytical purposes. Naive (unmodified) dsSNF7, treatment C2, was used as a comparison.
- SEQ ID Nos: 1-12 are provided above.
- ID 1 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA. NCBI Reference Sequence. NM 001305532 1 . 51-150 GGCAGCAACCATG GCGC TCAGCGATGCAGA TGTCCAAAAACAGATCAAGCATATGATGGCCTTCA TCGAGCAAGAGGCAAATGAAAAGGCCGAAGAAATC (SEQ ID NO: 13).
- ID 2 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 151-250 GATGCTAAGGCTGAGGAGGAGTTCAACATCGAGAAGGGGCGTCTGG
- ID 3 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 251-350 TGGAACTCCAGAAGAAGATCCAATCCTCCAACATGCTGAACCAGGCCCGTCTG
- ID 4 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 351-450 GACGCGCCGCCGCCTCGCCGAGGTGCCCAACGACCAGGGGCTCTACTCCGACC
- ID 5 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 451-550 ACCGTAACCCTCCGCGTGCGCGAGGCCGACAAGCCGCTGATCGACAGCCTGCT
- ID 6 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 551-650 CCTTGAAGGTGGACACGGAGCACTACCTGCCGGTGGGCACCTGCGGCGGGAT TGAGTTGGTCGCCGCTAGGGGCCGCATCAAGATCATCAACACCCTGGA (SEQ ID NO: 18).
- ID 7 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 651-750
- ID 8 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1501-1600
- ACATTCCTCTTTCATGTTTCCTGTCCTGGCAATCAAATGTTTCGGCT SEQ ID NO: 20.
- ID 9 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1601-1700 TGCTTTTAACAGTTCTATCGAAGAGCACCGTAGCTCTATAAATTACATAACGA
- ID 10 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1401-1500 ATACTGTTTACTATCGTGGACTTCCTGGGAATTATTTGATGCTGTAAGGTTTAT
- ID 11 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 51-166
- ID 25. 1001-1100 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA GGAACTTACACTAAAATACCTATGATCTTCGGATTTGTTGAAAACGAAGGAAC
- ID 25-4 XM_011559245.2
- ID 26 101-250 Plutella xylostella strain DBM1 Ac-S mitogen-activated protein kinase kinase kinase kinase 4 isoform XI (MAP4K4) mRNA, complete cds, alternatively spliced AATATAAAGTGCGTGATTTTACACATGTCGAATGTCATGAGTGAAAGGATCTT
- ID 27 901-1050 Plutella xylostella strain DBM1 Ac-S mitogen-activated protein kinase kinase kinase kinase 4 isoform XI (MAP4K4) mRNA, complete cds, alternatively spliced ATGGCCGAGAGTCAGCCGCCCCTGTGTGACCTTCACCCAATGAGAGCATTGTT
- ID 29. 301-400 Plutella xylostella prophenoloxidase 1 mRNA, complete cds GGAATGCGTGACGTGGAGGACCTGCAGTCCGTGTGTAGCTACTGCCAGCTCCG CATCAACCCCTACATGTTCAACTACTGCCTGTCGGTCGCCATGCTGC (SEQ ID NO: 44).
- ID 30. 401-500 Plutella xylostella prophenoloxidase 1 mRNA, complete cds ACAGACCAGACACGAAGGGCCTGTCGCCGCCGACGCTGGCGGAGACGTTCCC CGACAAGTTCATGGACCCCAAGGTGTTCCGCCGCGCGCGGGAGACCTC (SEQ ID NO: 45).
- ID 31 501-600 Plutella xylostella prophenoloxidase 1 mRNA, complete cds CACCACCACCGCGCCTGCTGGGGACAGGATGCCAGTCCTAATCCCGGTCAACTACA CGGCCTCCGACGCTGAGCCAGAACAACGCATCGCGTACTTCCGCGAA (SEQ ID NO: 46).
- ID 32 601-700 Plutella xylostella prophenoloxidase 1 mRNA, complete cds GACATCGGCATCAACCTGCACCACTGGCACTGGCACCTGGTGTACCCCTTCGA GGCGGCCGACCGCGCCGTGGTGGACAAGGACAGGCGCGGCGAGCTGCGC (SEQ ID NO: 47).
- ID 33 601-700 Plutella xylostella prophenoloxidase 1 mRNA, complete cds TGTACTACATGCACCAGCAGATCATCGCCAGATACAACGCAGAGCGTCTGTGC AACAACCTGGGCTTCGTGACGCGCTACAACGACTTCCGCGGGCCCAT (SEQ ID NO: 48).
- ID 34 801-900 Plutella xylostella prophenoloxidase 1 mRNA, complete cds CGCCGAGGGGTACTTCCCCAAGATGGACTCGCAGGTCGCCAGCAGGGCCTGG CCTCCTAGGTTCTCCGGCACCACGATCCGCGACCTGGACCGCCCCGTG (SEQ ID NO: 49).
- ID 35 901-1000 Plutella xylostella prophenol oxidase 1 mRNA, complete cds GACCAGATCCGCTCCGACGTGTCTGAGATGGAGACCTGGAGGGACCGCTTCAT CCAGGCCATCGACAGCGGCACTATTGTTCTGCCCAACGGCCGCACCC (SEQ ID NO: 50).
- ID 36 1001-1100 Plutella xylostella prophenoloxidase 1 mRNA, complete cds AGCGCCTCGACGAGGAGACCGGCATCGACGTGCTGGCCAACCTCATGGAGTC GTCCATCATCAGCCGCAACCGCGCCTACTACGGGGACCTGCACAACAT (SEQ ID NO: 51)
- ID 37 1101-1200 Plutella xylostella prophenoloxidase 1 mRNA, complete cds GGGGCATGTGTTCATCTCCTATGCGCACGACCCCGACCACCGGCACTTGGAAC AATTCGGCGTGATGGGAGACCCGGCCACGGCCATGAGGGACCCGATC (SEQ ID NO: 52).
- ID 38. 1201-1300 Plutella xylostella prophenoloxidase 1 mRNA, complete cds TTCTACCGCTGGCACGCGTACGTCGACGACATCTTCCAGAGATACAAGGCCAC ACTACCAGCCTACACCAGGGAGAGGTTGGACTTCCCAGGCATCCGCG (ID SEQ ID NO: 53).
- ID 40. 1301-1400 Plutella xylostella prophenoloxidase 1 mRNA, complete cds TCTCCTCCATCGCCATCTCGGGCCGCACTCCGAACCAGTTCTCGACGCAGTGG GAGCAGAGTTCAGTGAACCTGGCGCGCGGGCTGGACTTCATGCCGCG (SEQ ID NO: 54).
- ID 41 1401-1500 Plutella xylostella prophenoloxidase 1 mRNA, complete cds CGGCGCCGTGCTGGCGCGGTTCACGCATCTGCAGCATGACGAGTTTGAGTACA CCATCGAGTGCGACAACACAACCGGCCAAGCAGCCATGGGCACCGTC (SEQ ID NO: 55).
- ID 43 1601-1700 Plutella xylostella prophenol oxidase 1 mRNA, complete cds TGCGCCCCGGCAGCAACACCATCCGGCGTCGCAGCATCGACTCCTCAGTTACC ATCCCCTACGAGCGAACATTCCGGGACGAGTCCCAACACCCCGGAGA (SEQ ID NO: 57)
- ID 45 1801-1900 Plutella xylostella prophenol oxidase 1 mRNA, complete cds CTCTTCTGCATGATTACCAACTGGAATGAAGATCGGGTGGAGCAAGACACAGT AGGAACCTGCAACGACGCAGCCTCCTACTGCGGTATCCGGGACCGCC (SEQ ID NO: 59)
- ID 46 1901-2000 Plutella xylostella prophenol oxidase 1 mRNA, complete cds GCTACCCGGACCGCAAGCCTATGGGATTCCCCTTCGATAGACCAGCGCCATCT ACCGGCAGTTTGGGAGACTTCTTGACCCCCAACATGACTGTGCAGAA (SEQ ID NO: 60)
- ID 46-1 101-200 Plutella xylostella prophenol oxidase 1 mRNA, complete cds TTACAGTGTTCGTGGTATTTTAAGCCCAAACGCTAATCCAAGATGGCGGACAA AAACAACCTGCTGCTGTTCTTCGACCGCCCCACGGAGCCCTGCTTCA (SEQ ID NO: 61)
- ID 46-2 Plutella xylostella prophenol oxidase 1 mRNA, complete cds TCAGGTTTACTGATGCGGTCAGGCAGCGTCAGCAGCGGTAGAGTGTGGGAGG GAGACATGGTTCATACGTTACGAGAGTTCTAGCTAAACTTAAACACACAATGA AAGCTGACTGTAACTTTTGTAGATTTTGCCTTCAAAATGACAATTACTTAGGCA CTAATTAGGTTCTAATTTTGTATAATTCATTTTTAGCGACA (SEQ ID NO: 62) [00325] ID 46-3.
- ID 47 151-250 Plutella xylostella glutathione synthetase (Gss), mRNA CGACCCACGGCTCCAAGTCCGTGCTGACCCCCAACCTCGACGTGCTGACCCGC TCAGGAGTGTCCCTCCACCGCTACTACACCCACGCTCTCTGCTCGCC (SEQ ID NO: 64) [00327] ID 48.
- Gss Plutella xylostella glutathione synthetase
- Gss mRNA CGCCCGTACCGCTGTGCTCACCGGCAAATACGCCCACACCGTCGGTATGCAGG GTATGCCTCTGTCCAACGCTGAGGAGCGTGGTATCCCCCTAGAGGAG
- ID 49 351-450 Plutella xylostella glutathione synthetase (Gss), mRNA CGCCTGATCTCTCAGTACCTACAGGACGCTGGTTACAGGACCCAGATGGTCGG AAAGTGGCACGTCGGTCACGCCTTCTTCGAGCAGCTGCCCACTTACA (SEQ ID NO: 66)
- ID 50 451-550 Plutella xylostella glutathione synthetase (Gss), mRNA GAGGATTCGAGAACCACTTCGGTGTCCGCGGTGGATTCATCGACTACTACGAA TACAACGCTCAGGAGCA GCTTGACGGCAGGCCAGTCACTGGACTGTG (SEQ ID NO: 67)
- ID 51 551-650 Plutella xylostella glutathione synthetase (Gss), mRNA TCTGTTCGACGACCTGCAGCCCGACTGGACCACCGAGGGATACATCACCGACG TCTACACCGAGAAGTCCACCACCATCATTGAGAACCACAACGTCTCC (SEQ ID NO: 68)
- ID 52. 651-750 Plutella xylostella glutathione synthetase (Gss), mRNA GAGCCCCTGTACCTGCTGCTGACCCACCACGCTCCCCACAACGGCAACGAAGA CGCTTCCCTGCAGGCCCCTCCTGAAGAGGTCCGCGCCCAGAGGCACG (SEQ ID NO: 69)
- ID 53 751-850 Plutella xylostella glutathione synthetase (GSS), mRNA TCGAGCTCCACCCCAGACGTATCTTCGCCGCTATGGTTAAGAAACTGGACGAC AGTATCGGAGAAATCGTCGCTACCCTCGAGAAGAAGGGCATGCTCGA (SEQ ID NO: 70)
- ID 54 851-950 Plutella xylostella glutathione synthetase (Gss), mRNA GAACACCATCATCACCTTCTCCACTGACAACGGTGCCCCCACCGTCGGTCTTG GCGCCAACTCTGGTTCCAACTACCCCCTGAGAGGAGTCAAGAAGTCC (SEQ ID NO: 71)
- ID 54 951-1050 Plutella xylostella glutathione synthetase (Gss), mRNA CCCTGGGAGGGAGGTATCCGTGGTAACGCCATGATCTGGGCCGGTCCCGAGGT CGCCCCCGGAAACGCGTGGCGTGGAAAGGTTTACGACGGCAACATGC (SEQ ID NO: 72)
- ID 57. 1051-1150 Plutella xylostella glutathione synthetase (Gss), mRNA ACGCCGCTGACTGGGTCCCCACTCTGCTTGAGGCCATCGGTGAGAAGATCCCC GCCGGTCTGGACGGTATCCCCATGTGGAGCCACATCATCGAGAACAA (SEQ ID NO: 73)
- ID 58. 1151-1250 Plutella xylostella glutathione synthetase (Gss), mRNA GCCTTCTCCCCGTACCGAGATCTTCGAGATCGACGACTACTTCAACCACTCCTC TGTCACCCTCGGCCGCCACAAGCTCGTCAAGGGAACCATCGACGAG (SEQ ID NO: 74)
- ID 59. 1251-1350 Plutella xylostella glutathione synthetase (Gss), mRNA TCTCTCAGCAAGCACTACGGTGAAGACCTCCGTGGCATCATCGGAACTCCCCC AGACTACAAGCAGAAGCTGCGCGACAGCAAGGCATGGGAGTCTCTGG (SEQ ID NO: 75)
- ID 60. 1351-1450 Plutella xylostella glutathione synthetase (Gss), mRNA AGACCATCGGCATCCCTCTGGACGCTGACGTCATGGCTGACCGCGATGAGGCT ATCGTCACTTGCGGAAATGTCGTCCCCAAGCCTTGCAGTCCTTCTGC (SEQ ID NO: 76)
- ID 61. 1451-1550 Plutella xylostella glutathione synthetase (Gss), mRNA CGAGTCTTGGTGCCTGTACGACATCATCGAGGACCCTTGTGAGCTTCGTGACC TGTCTGAGGAGCTTCCTCAGCTGGCTCAGATCCTTCTGTACCGTCTG (SEQ ID NO: 77)
- ID 62. 1551-1650 Plutella xylostella glutathione synthetase (Gss), mRNA GAGCAGGAAGAGGCCAAGATCATCCCCAGGGAGGGCCAGTACGTCGCTGACC CCAAGTCTGCCCCCAAGTACTTCAACTACACCTGGGACGCGTACCTGT (SEQ ID NO: 78)
- ID 63. 1651-1750 Plutella xylostella glutathione synthetase (Gss), mRNA CCGTCGAACCCTACTCCGACTCCGAATAGACGAAGCTCAGCTCAAGCGGCGCA GTTCGCCGTGAAAGTTGTAAATGTTGATCCTGGCCTTAATTTCAGTA (SEQ ID NO: 79)
- ID 63-2 Plutella xylostella glutathione synthetase (Gss), mRNA
- AAAGTTGTAAATGTTGATCCTGGCCTTAATTTCAGTA (SEQ ID NO: 80) [00343] ID 64.
- ID 70. 651-751 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
- ID 71. 751-850 PREDICTED Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
- ID 73. 951-1050 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA AGTAGAGTGAAAGAACAACTAGCTAATAAAAATAAGGAAGGCCCAAGACAG
- ID 74. 1051-1150 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
- ID 75 201-350 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA AGGTCCATCAACACACGAAGCTATTCAAAAATTACGCGAGACCGAAGAATTG
- ID 76. 826-975 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA TAAGTGTTGGAAGCGAAACCGAATACATATGTATAGTCTCCGTTACACATCCA ACTACTTAGCTTATTCTAAGGCTGCGTCCCTGTAGGCAAACAGTATTCTCGTTA CTAGTCTATGGTAATTTGAGTAGAGTGAAAGAACAACTAGCTA (SEQ ID NO:
- ID 77. 826-975 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA TAAGTGTTGGAAGCGAAACCGAATACATATGTATAGTCTCCGTTACACATCCA ACTACTTAGCTTATTCTAAGGCTGCGTCCCTGTAGGCAAACAGTATTCTCGTTA CTAGTCTATGGTAATTTGAGTAGAGTGAAAGAACAACTAGCTA (SEQ ID NO:
- ID 78. 201-350, 826-975 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), purposed sequence mRNA AGGTCCATCAACACACGAAGCTATTCAAAAATTACGCGAGACCGAAGAATTG CTCATCAAAAAGCAGGACTTCCTGGAGAAGAAAATACAATTAGAAGTAGACA CAGCCAGGAAACATGGCACTAAGAACAAAAGAGCGGCCATCGCTGCTAAGTG TTGGAAGCGAAACCGAATACATATGTATAGTCTCCGTTACACATCCAACTACT TAGCTTATTCTAAGGCTGCGTCCCTGTAGGCAAACAGTATTCTCGTTACTAGTC TATGGTAATTTGAGTAGAGTGAAAGAACAACTAGCTA (SEQ ID NO: 95)
- ID 80 201-300 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GGTTATGTCTTCGCTGTGTCCGGTCCCGTGGTCACAGCGGAGAAGATGTCCGG CTCGGCCATGTACGAGCTGGTGCGCGTCGGCTACAACGAGCTGGTCG (SEQ ID NO: 97)
- ID 84 XM 038113977.1
- ID 85 XM_038113977.1
- ID 86 XM_038113977.1
- ID 88 XM_038113977.1
- ID 90 XM 038113977.1
- ID 97 300 BP Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, sequence mRNA
- ID 100 201-300 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
- IDs 106. 801-900 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds ACAGTCGTAACGGCGCCAAATCTTCGAGGAATGACATGGTGGATCTTATTTCC
- ID 107. 1201-1300 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
- ID 108. 1301-1400 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
- ID 109-2 AY971374.1
- ID 109-3 AY971374.1
- ID 109-11 1101-1158 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
- TCCCT (SEQ ID NO: 137)
- ID 109-11 1159-1216 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
- CGCTA (SEQ ID NO: 138) [00401] ID 109-12 1217-1274 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
- TTTGC (SEQ ID NO: 139)
- ID 109-13 1275-1333 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
- ID 109-14 1334-1390 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
- ID Pl. 101-250 Plutella xylostella cytochrome P450 6kl-like
- ID P2. 251-400 Plutella xylostella cytochrome P450 6kl-like
- ID P4 551-700 Plutella xylostella cytochrome P450 6kl-like
- ID P5. 701-850 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
- ID P6. 851-1000 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
- ID P7. 751-1050 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
- ID P8. 450-751 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
- ID 111. 301-400 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
- ID 114 MN480661.1
- ID 116 Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA NCBI Reference Sequence: XM 035591116.1
- AACTA SEQ ID NO: 1557
- ACATGGGTAAACCAAATGAAAGGCTGAGAAGGA SEQ ID NO: 160 [00423] ID 120. 101-200 PREDICTED: Spodoptera frugiperda cytochrome P450 dislike (LOCI 18273915), mRNA
- ID 121 201-300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2- like (LOCI 18273915), mRNA
- ID 125. 1201-1300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA
- ID 133 801-900 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
- ID. 1749-2049 Plutella xylostella strain DBM1 Ac-S ABC transporter subfamily H member 1 (ABCH1) mRNA, complete cds Sequence ID: KP260785.1 GACTACAACCCGAAGGTGGGCGACATCCCGATCGACTTCAAGGAGCCCATCT
- ID 138 1101-1250 Spodoptera frugiperda cytochrome P450 CYP9A58 mRNA, complete cds
- ID 143 351-450 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
- ID 144 451-550 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
- ID 150 801-950 PREDICTED: Spodoptera frugiperda charged multivesicular body protein 4b-like (LOCI 18279222), mRNA
- ID 158 101-200 Spodoptera frugiperda Dredd mRNA, complete cds AAATAATCATTTAAAACGAGAAAATGTTTTCTGTAGACTCAGTTACCACAACA CCACAAGGAATGGAAGTGGAAAATGTTATTGGAAACAGTGATATTAT (SEQ ID NO: 1).
- ID 160 301-400 Spodoptera frugiperda Dredd mRNA, complete cds CCACAACGTTTAATTGTTTTTCAACGCGTTTCCAACGATGCCTGTAACTCTATT AATCTTAACATGCTACACGAGTGGTTTCGATCTACTAAGCACAACC (SEQ ID NO: 195)
- ID 161 401-500 Spodoptera frugiperda Dredd mRNA, complete cds CCAACTGGAAACATCAATTTGTGGAAGCTCTCCTTATCTGTCAACTGTACAGT ATTGTCAGAAAACTTGGATTGAATGTCCCTACAGCACGCAAGTACTA (SEQ ID NO: 196)
- ID 162. 801-900 Spodoptera frugiperda Dredd mRNA, complete cds GATGAATATGTTGCAACACTCATTGAATGATGAGCACAAGCCAAGTGCCTCAG CTGCTACCAGTACTCCTATGATGAAACATATGAAAGTGGATGAAACA (SEQ ID NO: 197)
- ID 164 1001-1100 Spodoptera frugiperda Dredd mRNA, complete cds TGAAGTCTGATAGGAAGCTACTGGACAATGATAGCTATGAAATCAAGAGTAA CAAAAGGGTTGGAGTTTGTGTCATCATAAATCAGGAAACATTTTATCC (SEQ ID NO: 199)
- ID 165 1401-1500 Spodoptera frugiperda Dredd mRNA, complete cds TGATGCCAAAAAAATACACAACATGCCTAAACTTTTGATAGTTCAAGCATGCC AAGTTGATGAAAATACTCCCCAGATTGTAGTGGCTGACAGCCCAAGA (SEQ ID NO: 200)
- ID 166 1451-1550 Spodoptera frugiperda Dredd mRNA, complete cds GCCAAGTTGATGAAAATACTCCCCAGATTGTAGTGGCTGACAGCCCAAGAGAT TACAATTTAAGGAAATCTAACTTCCTTGTTTACTATGCCACTGCACC (SEQ ID NO: 201)
- ID 167 1501-1600 Spodoptera frugiperda Dredd mRNA, complete cds GATTACAATTTAAGGAAATCTAACTTCCTTGTTTACTATGCCACTGCACCTGAA CTAGAAGCTTACAGAAATGAAAAAAGAGGATCGATATTCATTCAGG (SEQ ID NO: 202)
- ID 169 Spodoptera frugiperda Dredd mRNA, complete cds GAATACTGATATGATGTCAGAAATTGAAAAAGAATTACAAGACAATCCCAGC GACTTGATATCCCTAGTGTTTCTTCTTTATGAAGTACCGGACACAGCATGAAGT CTGATAGGAAGCTACTGGACAATGATAGCTATGAAATCAAGAGTAACAAAAG GGTTGGAGTTTGTGTCATCATAAATCAGGAAACATTTTATCCTGATGCCAAAA AAATACACAACATGCCTAAACTTTTGATAGTTCAAGCATGCCAAGTTGATGAA AATACTCCCCAGATTGTAGTGGCTGACAGCCCAAGA (SEQ ID NO: 204) [00467] ID 170.
- ID 174 500-600 PREDICTED: Spodoptera frugiperda dual oxidase-like
- transcript variant XI (LOCI 18269141), transcript variant XI, mRNA
- CAA SEQ ID NO: 209
- ID 175. Spodoptera frugiperda dual oxidase-like
- transcript variant XI (LOCI 18269141), transcript variant XI, mRNA
- ID 177 2600-2700 PREDICTED: Spodoptera frugiperda dual oxidase-like
- transcript variant XI (LOCI 18269141), transcript variant XI, mRNA
- transcript variant XI (LOCI 18269141), transcript variant XI, mRNA
- transcript variant XI (LOCI 18269141), transcript variant XI, mRNA
- transcript variant XI mRNA TATCAGGTCAATGGGTACGGTTATCTTGTACGGCGTTCAAGAAAGAAGAGTTC CACTCGTTCACATTAACCTCAGCTCCTCACGAGAACTTCTTATCGTGT (SEQ ID NO: 215)
- ID 185. 301-400 PREDICTED Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA
- ID 187. 501-600 PREDICTED Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA
- ID 190 Spodoptera frugiperda glutathione S-transferase 1-like
- ID 195 [00494] ID 195. 401-500 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA GAAATACTAAGTCCAACGGATGATCTGCAAGTAAGAAGCGGGAAATATCAGC TCAATGATGGGCTCGTGGGTGAAGAGCCGATGCCACTAGATGCTGTTA (SEQ ID NO: 231)
- ID 196 1901-2000 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA AAGGAGATTACCATCAGACCCCAGCTTGAATACATAGATATTATCGAAATGGG CGTGGCTAACACTGGAGAATATGTGATCAATCCCCAAAACTTTAGGA (SEQ ID NO: 232)
- LOCI 18263801 LOCI 18263801
- transcript variant XI mRNA AAGTCGGACGTTATACAAAGAACGATGAGTGTGCTGAGGGGTTTTTAGTGAG AAATTGTGAAAAATAGAAAGAGTGAAGATGTCGGTAAAAGCGAGCGTT (SEQ ID NO: 238)
- ID 204 401-500 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA GGTTAAACAAAAAGGGTAAAAATGGCCTTCAAAGGATTCTGTGGCGAAGTGA TCGGGTTTTTCCTGGCTGTGGGTTTTTGCATCATATGTCCGGAATATG (SEQ ID NO: 239)
- ID 205 701-800 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA AACTGCCGAGTCAAGAATTTAGGAAATAAAACGTTAAACATGCAAGTATCGT GGGTACGGCATAGAGACATCCATCTGCTGACAGTCGGCCGGTACACAT (SEQ ID NO: 240)
- ID 206 801-900 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA ACACGAGCGATCAAAGGTTTAGAGCTATTCACTTACCGCACTCCGAGGACTGG ACTTTACAGATCAAGTATCCGCAACACAGGGATTCGGGAATTTATGA (SEQ ID NO: 241)
- ID 207. 1351-1450 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA GTTGGTTGCCTGCCTGTTCATCGCACTCTCTTGACACATACGGATCTTTAACTA AATGTAAATACAAGGATTTTATCAACATCAGCTGCTTATATTGACA (SEQ ID NO: 242)
- ID 211 Spodoptera frugiperda uncharacterized LOC 118263801 (LOCI 18263801), transcript variant XI, mRNA AAGTCGGACGTTATACAAAGAACGATGAGTGTGCTGAGGGGTTTTTAGTGAG AAATTGTGAAAAATAGAAAGAGTGAAGATGTCGGTAAAAGCGAGCGTTGGTT AAACAAAAAGGGTAAAAATGGCCTTCAAAGGATTCTGTGGCGAAGTGATCGG GTTTTTCCTGGCTGTGGGTTTTTGCATCATATGTCCGGAATATG (SEQ ID NO: 243) [00507] ID 212. 401-500 Spodoptera frugiperda V-ATPase subunit A mRNA, complete cds AATTCAACCCCTTGAATGTTAAGGTCGGCTCCCACATCACCGGAGGAGACTTG
- ID 214 1501-1600 Spodoptera frugiperda V-ATPase subunit A mRNA, complete cds
- ID 218 100-159, 1-60, Spodoptera frugiperda V-type proton ATPase catalytic subunit A (LOCI 18267501), transcript variant XI, X2, X3, and X4
- ID 220 301-400 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
- ID 222 1101-1200 Spodoptera frugiperda cytochrome P450 CY321 A8 mRNA, complete cds
- AAGAAGGACGGAAAAGTATTGTCCCGTATACCTAC (SEQ ID NO: 254) [00518] ID. 201-500 Spodoptera frugiperda cytochrome P450 CYP6AE44 mRNA, complete cds Sequence ID: MN480661.1
- ID 25-2 351-500 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA CCAACGTCTACACACCGGCCATTGATCCAGAAAAGAAATACCCAGTAATGGTT TGGATTAAAGGGTCCGAGTTTGAGAAAACTAAGGGACCTGAACTATCTTTTAG AAATCTTATTGAAAAAGAAGTAATAGTCGTGTCTCTAAACTTCATAGATGATG AAAAGTTTCTAGAAAAATCACCTTTTAGTACGCTAACTGAAGGAACTTACACT AAAATACCTATGATCTTCGGATTTGTTGAAAACGAAGGAACAATACGTTTTGA TGAGGCACTAGAAGCTGATTGGCTAACAAAGATGG (SEQ ID NO: 260).
- Diabrotica virgifera virgifera charged multivesicular body protein 4b (LOCI 14337301), mRNA Sequence ID: XM_028287710.1 gcaaagaaaaatgcgtcgaaaataaaagagttgcactccaagccctcaaaaagaagaaacgattggaaaagacccaactaca aatagatggaacccttacaactattgaaatgcagagggaagccctcgaaggagctagcacaaatactgctgtattagattctatgaa aatgctgcagatgcccttaagaaagctcataagaatttgaatgtagatgatgttcacgatatcatggat (SEQ ID NO: 264)
- ID. 87-386 PREDICTED: Plutella xylostella cytokine receptor-like (LOCI 05380229), mRNA Sequence ID: XM_011549746.3 Domeless: CTTCAAATTATTGTAGTGATGATCATACGGAAGCCGATTTGATAGAATTTTAA TTCACATACGATACTAGCCTATATAACAGTCGATTTGTGCAGTCGGTCAGTGA GTGTTGTTTAGTGAGTCGTGCAACTCGTACCCTGCATGACTTTGGACTCCGATG CGCCATTTGGCCTCAGCTATAAAAACAAGGAATGTGTTTATCGTCTCTCCCGA TTTCCCAAACGACTTTAGCTTGCAGTGATGGTGATAAGTGAACTTTAGTGA
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